Integrated sensor for multi-dimensional signal analysis

ABSTRACT

Some aspects relate to an integrated circuit, comprising at least one photodetection region configured to generate charge carriers responsive to incident photons emitted from a sample, at least one charge storage region configured to receive the charge carriers from the photodetection region, and at least one controller configured to obtain information about the incident photons, the information comprising at least one member selected from the group comprising pulse duration and interpulse duration and at least one member selected from the group comprising wavelength information, luminescence lifetime information, and intensity information. In some embodiments, the information comprises at least three, four, and/or five members selected from the group comprising wavelength information, luminescence lifetime information, intensity information, pulse duration information, and interpulse duration information. In some embodiments, the information obtained may be used to identify the sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/190,331, entitled “INTEGRATED SENSOR FOR MULTI-DIMENSIONAL SIGNALANALYSIS”, filed on Mar. 2, 2021, which claims priority to U.S.provisional patent application Ser. No. 62/984,229, entitled “INTEGRATEDSENSOR FOR MULTI-DIMENSIONAL SIGNAL ANALYSIS”, filed Mar. 2, 2020 underAttorney Docket No. R0708.70090US00, which is incorporated by referencein its entirety herein.

FIELD OF THE DISCLOSURE

The present disclosure relates to integrated devices and relatedinstruments that can perform massively-parallel analyses of samples byproviding short optical pulses to tens of thousands of sample wells ormore simultaneously and receiving fluorescent signals from the samplewells for sample analyses. The instruments may be useful forpoint-of-care genetic sequencing and for personalized medicine.

BACKGROUND

Photodetectors are used to detect light in a variety of applications.Integrated photodetectors have been developed that produce an electricalsignal indicative of the intensity of incident light. Integratedphotodetectors for imaging applications include an array of pixels todetect the intensity of light received from across a scene. Examples ofintegrated photodetectors include charge coupled devices (CCDs) andComplementary Metal Oxide Semiconductor (CMOS) image sensors.

Instruments that are capable of massively-parallel analyses ofbiological or chemical samples are typically limited to laboratorysettings because of several factors that can include their large size,lack of portability, requirement of a skilled technician to operate theinstrument, power need, need for a controlled operating environment, andcost. When a sample is to be analyzed using such equipment, a commonparadigm is to extract a sample at a point of care or in the field, sendthe sample to the lab and wait for results of the analysis. The waittime for results can range from hours to days.

SUMMARY OF THE DISCLOSURE

Some aspects of the present disclosure relate to an integrated circuit,comprising: at least one photodetection region configured to generatecharge carriers responsive to incident photons emitted from a sample; atleast one charge storage region configured to receive the chargecarriers from the photodetection region; and at least one componentconfigured to obtain information about the incident photons, theinformation comprising at least one of pulse duration information and/orinterpulse duration information and at least one member selected from agroup comprising wavelength information, luminescence lifetimeinformation, and intensity information.

Some aspects of the disclosure relate to an integrated circuit,comprising: at least one photodetection region configured to generatecharge carriers responsive to incident photons emitted from a sample; atleast one charge storage region configured to receive the chargecarriers from the photodetection region; and at least one componentconfigured to obtain information about the incident photons, theinformation comprising at least three of wavelength information,luminescence lifetime information, intensity information, pulse durationinformation, and interpulse duration information.

Some aspects of the disclosure relate to an integrated circuit,comprising: at least one photodetection region configured to generatecharge carriers responsive to incident photons emitted from a sample; atleast one charge storage region configured to receive the chargecarriers from the photodetection region; and at least one componentconfigured to obtain information about the incident photons, theinformation comprising interpulse duration information and at least onemember selected from a group comprising wavelength information,luminescence lifetime information, intensity information, and pulseduration information.

Some aspects of the disclosure relate to an integrated circuit,comprising: at least one photodetection region configured to generatecharge carriers responsive to incident photons emitted from a sample; atleast one charge storage region configured to receive the chargecarriers from the photodetection region; and at least one componentconfigured to obtain information about the incident photons, theinformation comprising at least three members selected from s groupcomprising wavelength information, luminescence lifetime information,intensity information, pulse duration information, and interpulseduration information.

Some aspects of the disclosure relate to an integrated circuit,comprising: at least one photodetection region configured to generatecharge carriers responsive to incident photons emitted from a sample; atleast one charge storage region configured to receive the chargecarriers from the photodetection region; and at least one componentconfigured to obtain information about the incident photons, theinformation comprising at least four members selected from s groupcomprising wavelength information, luminescence lifetime information,intensity information, pulse duration information, and interpulseduration information.

Some aspects of the disclosure relate to an integrated circuit,comprising: at least one photodetection region configured to generatecharge carriers responsive to incident photons emitted from a sample; atleast one charge storage region configured to receive the chargecarriers from the photodetection region; and at least one componentconfigured to obtain information about the incident photons, theinformation comprising wavelength information, luminescence lifetimeinformation, intensity information, pulse duration information, andinterpulse duration information.

Some aspects of the disclosure relate to a device that determinesinformation about a sample which emits emission light in response toexcitation light, the information comprising at least one memberselected from a group comprising pulse duration and interpulse durationcharacteristics of the emission light and at least one member selectedfrom a group comprising wavelength, intensity, and luminescence lifetimecharacteristics of the emission light.

Some aspects of the disclosure relate to a device, comprising: a samplewell that receives a sample which emits emission light in response toexcitation light; a photodetector that produces charge carriers inresponse to the emission light; and a component that determines at leasttwo members selected from a group comprising wavelength, intensity,luminescence lifetime, pulse duration, and interpulse durationcharacteristics of the emission light based at least in part on one ormore characteristics of the charge carriers.

Some aspects of the disclosure relate to a method, comprising: excitinga sample with excitation light; and determining at least three membersselected from a group comprising wavelength, intensity, lifetime, pulseduration and interpulse duration characteristics of light emitted by thesample in response to the excitation light.

Some aspects of the disclosure relate to a method, comprising: excitinga sample with excitation light; determining pulse durationcharacteristics of light emitted by the sample in response to theexcitation light; and determining at least one member selected from agroup comprising wavelength, intensity, lifetime, and interpulseduration characteristics of the light emitted by the sample in responseto the excitation light.

Some aspects of the disclosure relate to a method, comprising: excitinga sample with excitation light; determining interpulse durationcharacteristics of light emitted by the sample in response to theexcitation light; and determining at least one member selected from agroup comprising wavelength, intensity, lifetime, and pulse durationcharacteristics of the light emitted by the sample in response to theexcitation light.

Some aspects of the disclosure relate to a method, comprising:determining information about a sample that emits emission light inresponse to excitation light based on at least three members selectedfrom a group comprising wavelength, intensity, lifetime, pulse durationand interpulse duration characteristics of the emission light.

Some aspects of the disclosure relate to a device, comprising: aplurality of sample wells, each sample well configured to receive asample that, in response to excitation light, emits emission light; awaveguide that directs the excitation light to the plurality of samplewells; a plurality of sensors, each sensor of the plurality of sensorsconfigured to generate charge carriers in response to the emissionlight; and one or more processors configured to determine at least onemember selected from a group comprising pulse duration and interpulseduration characteristics of the emission light and one member selectedfrom a group comprising wavelength, lifetime, and intensitycharacteristics of the emission light from one or more sample wells ofthe plurality of sample wells and to determine information about thesample based at least in part on the at least two characteristics.

Some aspects of the disclosure relate to a device, comprising: aplurality of sample wells, each sample well configured to receive asample that, in response to excitation light, emits emission light; awaveguide that directs the excitation light to the plurality of samplewells; a plurality of sensors, each sensor of the plurality of sensorsconfigured to generate charge carriers in response to the emissionlight; and one or more processors configured to determine at least threemembers selected from a group comprising wavelength, lifetime,intensity, pulse duration and interpulse duration characteristics of theemission light from one or more sample wells of the plurality of samplewells and to determine information about the sample based at least inpart on the at least two characteristics.

Some aspects of the disclosure relate to a device, comprising: at leastone photodetection region configured to generate charge carriersresponsive to incident photons emitted from a sample; and at least onecomponent configured to obtain information about the incident photons,the information comprising any of three, four, or five characteristicsselected from a group comprising wavelength information, luminescencelifetime information, intensity information, pulse duration information,and interpulse duration information.

Some aspects of the disclosure relate to an integrated circuitcomprising: at least one photodetection region configured to generatecharge carriers responsive to incident photons emitted from a sample; atleast one component configured to obtain information about the incidentphotons, the information comprising any of three, four, or five membersselected from a group comprising pulse duration information, interpulseduration information, wavelength information, luminescence lifetimeinformation, and intensity information, wherein: the at least onecomponent comprises a plurality of components, each one of the pluralityof components being configured to obtain a different one of the three,four, or five members selected from the group comprising pulse durationinformation, interpulse duration information, wavelength information,luminescence lifetime information, and intensity information.

Some aspects of the disclosure relate to an integrated circuitcomprising: at least one photodetection region configured to generatecharge carriers responsive to incident photons emitted from a sample; atleast one component configured to obtain information about the incidentphotons, the information comprising any of three, four, or fivecharacteristics selected from a group comprising pulse durationinformation, interpulse duration information, wavelength information,luminescence lifetime information, and intensity information, wherein:the at least one component comprises a first component being configuredto obtain each of the three, four, or five members selected from thegroup comprising pulse duration information, interpulse durationinformation, wavelength information, luminescence lifetime information,and intensity information.

Some aspects of the disclosure relate to an integrated circuitcomprising: at least one photodetection region configured to generatecharge carriers responsive to incident photons emitted from a sample; atleast one component configured to obtain information about the incidentphotons, the information comprising any of three, four, or fivecharacteristics selected from a group comprising pulse durationinformation, interpulse duration information, wavelength information,luminescence lifetime information, and intensity information, the atleast one component comprising: a first component being configured toobtain at least two of the members selected from the group comprisingpulse duration information, interpulse duration information, wavelengthinformation, luminescence lifetime information, and intensityinformation; and a second component being configured to obtain at leastone of the members selected from the group comprising pulse durationinformation, interpulse duration information, wavelength information,luminescence lifetime information and intensity information; the atleast one member obtained by the second component being different thanthe at least two members obtained by the first component.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1-1A is a schematic of an integrated device, according to someembodiments.

FIG. 1-1B is a schematic of a pixel of an integrated device, accordingto some embodiments.

FIG. 1-1C is a circuit diagram of the pixel of FIG. 1-1B, according tosome embodiments.

FIG. 1-1D is a plan view of the pixel of FIGS. 1-1B and 1-1C, accordingto some embodiments.

FIG. 1-2A depicts emission probability curves for two differentfluorophores having different decay characteristics, according to someembodiments.

FIG. 1-2B depicts time-binning detection of fluorescent emission,according to some embodiments.

FIG. 1-2C depicts a time-binning photodetector, according to someembodiments.

FIG. 1-2D depicts a pulsed excitation and time-binned detection offluorescent emission from a sample, according to some embodiments.

FIG. 1-2E depicts a histogram of accumulated fluorescent photon countsin various time bins after a repeated pulsed excitation of a sample,according to some embodiments.

FIG. 2-1 is a side view of an exemplary pixel having a time-gated chargestorage region and a direct-excitation charge storage region configuredto receive incident photons from a light source via a photodetectionregion, according to some embodiments.

FIG. 2-2 is a side view of an alternative exemplary pixel having atime-gated charge storage region and a direct-excitation charge storageregion configured to receive incident photons from a light source via aphotodetection region, according to some embodiments.

FIG. 2-3 is a side view of an exemplary pixel having a time-gated chargestorage region, a direct-excitation charge storage region configured toreceive incident photons from a light source, and a photodetectionregion configured to receive at least some of the incident photons viathe direct-excitation charge storage region, according to someembodiments.

FIG. 2-4 is a side view of an exemplary pixel having two photodetectionregions of different depths and two time-gated charge storage regions,according to some embodiments.

FIG. 2-5 is a side view of an exemplary pixel having threephotodetection regions of different depths, according to someembodiments.

FIG. 3-1A is a top view of an exemplary pixel having one photodetectionregion and four charge storage regions, according to some embodiments.

FIG. 3-1B is a cross-sectional view of a portion of the pixel of FIG.3-1A, according to some embodiments.

FIG. 3-2A is a top view of an exemplary pixel having one photodetectionregion and six charge storage regions, according to some embodiments.

FIG. 3-2B is a cross-sectional view of a portion of the pixel of FIG.3-2A, according to some embodiments.

FIG. 3-3A is a top view of an exemplary pixel having one photodetectionregion and two charge storage regions, according to some embodiments.

FIG. 3-3B is a cross-sectional view of a portion of the pixel of FIG.3-3A, according to some embodiments.

FIG. 3-3C is an alternate cross-sectional view of a portion of the pixelof FIG. 3-3A, according to some embodiments.

FIG. 3-4 is a side view of an example pixel having two charge storageregions with one of the charge storage regions having optical shielding,according to some embodiments.

FIG. 4-1 is a side view of an exemplary pixel having an optical sortingelement, two photodetection regions, and two time-gated charge storageregions, according to some embodiments.

FIG. 4-2 is a side view of an alternative exemplary pixel having anoptical sorting element, two photodetection regions, and two time-gatedcharge storage regions, according to some embodiments.

FIGS. 4-3A-E show non-limiting examples of characteristic pulse patternsfor sample molecules under analysis, according to some embodiments.

FIG. 4-4 shows one example of data collected for a multi-dimensionaldiscrimination technique, according to some embodiments.

FIG. 5-1A is a block diagram of an integrated device and an instrument,according to some embodiments.

FIG. 5-1B is a schematic of an apparatus including an integrated device,according to some embodiments.

FIG. 5-1C is a block diagram depiction of an analytical instrument thatincludes a compact mode-locked laser module, according to someembodiments.

FIG. 5-1D depicts a compact mode-locked laser module incorporated intoan analytical instrument, according to some embodiments.

FIG. 5-2 depicts a train of optical pulses, according to someembodiments.

FIG. 5-3 depicts an example of parallel reaction chambers that can beexcited optically by a pulsed laser via one or more waveguides accordingto some embodiments.

FIG. 5-4 illustrates optical excitation of a reaction chamber from awaveguide, according to some embodiments.

FIG. 5-5 depicts further details of an integrated reaction chamber,optical waveguide, and time-binning photodetector, according to someembodiments.

FIG. 5-6 depicts an example of a biological reaction that can occurwithin a reaction chamber, according to some embodiments.

FIG. 5-7 depicts emission probability curves for two differentfluorophores having different decay characteristics according to someembodiments.

FIG. 5-8 depicts time-binning detection of fluorescent emission,according to some embodiments.

FIG. 5-9 depicts a time-binning photodetector, according to someembodiments.

FIG. 5-10A depicts pulsed excitation and time-binned detection offluorescent emission from a sample, according to some embodiments.

FIG. 5-10B depicts a histogram of accumulated fluorescent photon countsin various time bins after repeated pulsed excitation of a sample,according to some embodiments.

FIG. 5-11A-5-11D depict different histograms that may correspond to fournucleotides (T, A, C, G) or nucleotide analogs, according to someembodiments.

FIG. 5-12 illustrates an example graph illustrating a three-dimensionaldiscrimination technique for identifying amino acids

FIG. 5-13 is a flow diagram illustrating a method of sequencing alabeled polypeptide by Edman degradation according to some embodiments.

FIG. 5-14 includes a flow diagram illustrating a method of sequencing inwhich discrete binding events give rise to signal pulses of a signaloutput, and a graph illustrating the signal output according to someembodiments.

The features and advantages of the present invention will become moreapparent from the detailed description set forth below when taken inconjunction with the drawings. When describing embodiments in referenceto the drawings, directional references (“above,” “below,” “top,”“bottom,” “left,” “right,” “horizontal,” “vertical,” etc.) may be used.Such references are intended merely as an aid to the reader viewing thedrawings in a normal orientation. These directional references are notintended to describe a preferred or only orientation of features of anembodied device. A device may be embodied using other orientations.

DETAILED DESCRIPTION

I. Introduction

Aspects of the present disclosure relate to integrated devices,instruments and related systems capable of analyzing samples inparallel, including identification of single molecules and nucleic acidsequencing. Such an instrument may be compact, easy to carry, and easyto operate, allowing a physician or other provider to readily use theinstrument and transport the instrument to a desired location where caremay be needed. Analysis of a sample may include labeling the sample withone or more fluorescent markers, which may be used to detect the sampleand/or identify single molecules of the sample (e.g., individualnucleotide identification as part of nucleic acid sequencing). Afluorescent marker may become excited in response to illuminating thefluorescent marker with excitation light (e.g., light having acharacteristic wavelength that may excite the fluorescent marker to anexcited state) and, if the fluorescent marker becomes excited, emitemission light (e.g., light having a characteristic wavelength emittedby the fluorescent marker by returning to a ground state from an excitedstate). Detection of the emission light may allow for identification ofthe fluorescent marker, and thus, the sample or a molecule of the samplelabeled by the fluorescent marker. According to some embodiments, theinstrument may be capable of massively-parallel sample analyses and maybe configured to handle tens of thousands of samples or moresimultaneously.

The inventors have recognized and appreciated that an integrated device,having sample wells configured to receive the sample and integratedoptics formed on the integrated device, and an instrument configured tointerface with the integrated device may be used to achieve analysis ofthis number of samples. The instrument may include one or moreexcitation light sources, and the integrated device may interface withthe instrument such that the excitation light is delivered to the samplewells using integrated optical components (e.g., waveguides, opticalcouplers, optical splitters) formed on the integrated device. Theoptical components may improve the uniformity of illumination across thesample wells of the integrated device and may reduce a large number ofexternal optical components that might otherwise be needed. Furthermore,the inventors have recognized and appreciated that integratingphotodetectors (e.g., photodiodes) on the integrated device may improvedetection efficiency of fluorescent emissions from the sample wells andreduce the number of light-collection components that might otherwise beneeded.

The inventors have further recognized that certain characteristics offluorescent emissions from the sample wells may be measured and analyzedfor use in a number of applications. For example, certaincharacteristics of emitted light may enable identification of the samplebeing analyzed (e.g., identification of a fluorescent marker) which canfacilitate genetic sequencing applications, such as DNA, RNA, and/orprotein sequencing. Multiple characteristics of emission light may beobtained, including information regarding intensity, wavelength,lifetime, pulse duration, interpulse duration and any combinationthereof, to enable multi-dimensional discrimination techniques foranalyzing a chemical or biological sample. In some embodiments, thedevice is configured to obtain measurements for characteristics ofemitted light to enable techniques for 2-D, 3-D, 4-D, and 5-Ddiscrimination of one or more samples under analysis.

For example, the inventors have developed techniques for obtainingspectral information such as wavelength of the incident light emittedfrom a sample well. For instance, in some aspects, a pixel may includeone or more charge storage regions configured to receive charge carriersgenerated responsive to incident photons from a light source, withcharge carriers stored in the charge storage region(s) indicative ofspectral and timing information. In one example, two charge storageregions may receive charge carriers generated responsive to incidentlight at different wavelengths, such that a difference in power spectraldensity of the incident light is indicated in the accumulated charge inthe charge storage regions. Alternatively or additionally, in someaspects, a pixel may include regions having different depths, eachconfigured to generate charge carriers responsive to incident photons.For instance, in one example, the pixel may include two or morephotodetection regions having different depths (e.g., along the opticalaxis) such that charge carriers are generated in the differentphotodetection regions responsive to incident photons of differentwavelengths. Alternatively or additionally, in some aspects, a pixel mayinclude multiple charge storage regions having different depths, and oneor more of the charge storage regions may be configured to receive theincident photons and generate charge carriers therein. Another of thecharge storage regions may be configured to receive charge carriersgenerated in the photodetection region(s) of the pixel. In some aspects,a pixel may alternatively or additionally include an optical sortingelement configured to direct at least some incident photons to onecharge storage region and other incident photons to another chargestorage region. For instance, in one example, the optical sortingelement may include an at least partially refractive, diffractive,scattering, and/or plasmonic element. The inventors have recognized thatwavelength information may be used as one degree of discrimination insome embodiments of 2-D, 3-D, 4-D and/or 5-D discrimination sampleanalysis techniques.

In addition, the inventors have developed methods for obtaining,separately and in any combination, lifetime, pulse duration, interpulseduration, and intensity information for a sample under analysis. Inparticular, time-gating techniques may be used to obtain measurements offluorescence lifetime, pulse width/duration, and/or interpulse durationof an emission from a sample under analysis. In some embodiments, one ormore measurements for intensity of emission light are obtained bycollecting and quantifying charge carriers generated by incident photonsin one or more charge storage regions. The inventors have recognizedthat such fluorescence lifetime, pulse duration, interpulse duration,and/or intensity information may be used as degrees of discrimination insome embodiments of 2-D, 3-D, 4-D and/or 5-D discrimination sampleanalysis techniques in addition or alternative to wavelengthinformation. For example, in 2-D discrimination techniques,discrimination of a sample may be based on any two types of informationused in combination, such as wavelength and intensity, lifetime andintensity, etc. For 3-D discrimination techniques, discrimination of asample may be performed based on any three types of information used incombination, such as wavelength, lifetime, and intensity, etc. Likewise,4-D discrimination techniques may be performed based on any four typesof information used in combination and 5-D discrimination techniques maybe performed based on any five types of information used in combination.

Thus, the inventors have recognized that the techniques described hereinfor obtaining wavelength, lifetime, pulse duration, interpulse duration,and intensity information and/or any other suitable characteristic ofemission light from a sample may be used to facilitate multi-dimensionalanalysis of a biological or chemical sample using any combination ofcharacteristics described herein. In some embodiments, themulti-dimensional analysis may be used for identifying the particularsample from which emission light is collected and analyzed, for example,identifying a particular amino acid or nucleotide. The inventors haverecognized that multi-dimensional analysis of a sample can provide formore accurate identification of a molecule as opposed to singledimensional analysis. In addition, techniques using more dimensions canprovide for more accurate identification of a molecule compared totechniques which use fewer dimensions for analysis.

In some embodiments, a two-dimensional discrimination technique is usedfor analyzing and identifying a sample based on characteristics ofemission light associated with the sample. Any suitable grouping ofcharacteristics may be used in such two-dimensional techniques, forexample, lifetime and wavelength information, and/or wavelength andintensity information. In some embodiments, a three-dimensionaldiscrimination technique is used for analyzing and identifying a sample,for example, using wavelength, lifetime, and intensity information,using wavelength, pulse duration, interpulse duration, and lifetimeinformation, and/or any other suitable grouping of characteristics. Insome embodiments, a four-dimensional discrimination technique is usedfor analyzing and identifying a sample, for example, using wavelength,lifetime, intensity, pulse duration and interpulse duration informationof collected emission light associated with a sample under analysis. Insome embodiments, the integrated device is configured for massivelyparallel sample analysis and thus the multi-dimensional analysistechniques can be used for analyzing and identifying a high volume ofsamples at a time.

It should be appreciated that integrated devices described herein mayincorporate any or all techniques described herein alone or incombination.

II. Integrated Device Overview

The multi-dimensional signal analysis techniques described herein may,in some embodiments, be implemented using an integrated device, such asintegrated device 1-102 shown in FIG. 1-1A. A cross-sectional schematicof integrated device 1-102 illustrating a row of pixels 1-112 is shownin FIG. 1-1A. Integrated device 1-102 may include coupling region 1-201,routing region 1-202, and pixel region 1-203. Pixel region 1-203 mayinclude a plurality of pixels 1-112 having sample wells 1-108 positionedon a surface at a location separate from coupling region 1-201, which iswhere excitation light (shown as the dashed arrow) couples to integrateddevice 1-102. Sample wells 1-108 may be formed through metal layer(s)1-106. One pixel 1-112, illustrated by the dotted rectangle, is a regionof integrated device 1-102 that includes a sample well 1-108 and aphotodetection region having one or more photodetectors 1-110.

FIG. 1-1A illustrates the path of excitation (shown in dashed lines) bycoupling a beam of excitation light to coupling region 1-201 and tosample wells 1-108. The row of sample wells 1-108 shown in FIG. 1-1A maybe positioned to optically couple with waveguide 1-220. Excitation lightmay illuminate a sample located within a sample well. The sample mayreach an excited state in response to being illuminated by theexcitation light. When a sample is in an excited state, the sample mayemit emission light, which may be detected by one or more photodetectorsassociated with the sample well. FIG. 1-1A schematically illustrates anoptical axis of emission light OPT from a sample well 1-108 tophotodetector(s) 1-110 of pixel 1-112. The photodetector(s) 1-110 ofpixel 1-112 may be configured and positioned to detect emission lightfrom sample well 1-108. Examples of suitable photodetectors aredescribed in U.S. patent application Ser. No. 14/821,656 titled“INTEGRATED DEVICE FOR TEMPORAL BINNING OF RECEIVED PHOTONS,” filed Aug.7, 2015 under Attorney Docket No. R0708.70002US02 which is herebyincorporated by reference in its entirety. For an individual pixel1-112, a sample well 1-108 and its respective photodetector(s) 1-110 maybe aligned along a common axis (along the y-direction shown in FIG.1-1A). In this manner, the photodetector(s) may overlap with the samplewell within a pixel 1-112.

The directionality of the emission light from a sample well 1-108 maydepend on the positioning of the sample in the sample well 1-108relative to metal layer(s) 1-106 because metal layer(s) 1-106 may act toreflect emission light. In this manner, a distance between metallayer(s) 1-106 and a fluorescent marker positioned in a sample well1-108 may impact the efficiency of photodetector(s) 1-110, that are inthe same pixel as the sample well, to detect the light emitted by thefluorescent marker. The distance between metal layer(s) 1-106 and thebottom surface of a sample well 1-106, which is proximate to where asample may be positioned during operation, may be in the range of 100 nmto 500 nm, or any value or range of values in that range. In someembodiments the distance between metal layer(s) 1-106 and the bottomsurface of a sample well 1-106 is approximately 300 nm.

The distance between the sample and the photodetector(s) may also impactefficiency in detecting emission light. By decreasing the distance lighthas to travel between the sample and the photodetector(s), detectionefficiency of emission light may be improved. In addition, smallerdistances between the sample and the photodetector(s) may allow forpixels that occupy a smaller area footprint of the integrated device,which can allow for a higher number of pixels to be included in theintegrated device. The distance between the bottom surface of a samplewell 1-106 and photodetector(s) may be in the range of 5 μm to 15 μm, orany value or range of values in that range. It should be appreciatedthat, in some embodiments, emission light may be provided through othermeans than an excitation light source and a sample well. Accordingly,some embodiments may not include sample well 1-108.

Photonic structure(s) 1-230 may be positioned between sample wells 1-108and photodetectors 1-110 and configured to reduce or prevent excitationlight from reaching photodetectors 1-110, which may otherwise contributeto signal noise in detecting emission light. As shown in FIG. 1-1A, theone or more photonic structures 1-230 may be positioned betweenwaveguide 1-220 and photodetectors 1-110. Photonic structure(s) 1-230may include one or more optical rejection photonic structures includinga spectral filter, a polarization filter, and a spatial filter. Photonicstructure(s) 1-230 may be positioned to align with individual samplewells 1-108 and their respective photodetector(s) 1-110 along a commonaxis. Metal layers 1-240, which may act as a circuitry for integrateddevice 1-102, may also act as a spatial filter, or polarization filter,in accordance with some embodiments. In such embodiments, one or moremetal layers 1-240 may be positioned to block some or all excitationlight from reaching photodetector(s) 1-110.

Coupling region 1-201 may include one or more optical componentsconfigured to couple excitation light from an external excitationsource. Coupling region 1-201 may include grating coupler 1-216positioned to receive some or all of a beam of excitation light.Examples of suitable grating couplers are described in U.S. patentapplication Ser. No. 15/844,403 titled “OPTICAL COUPLER AND WAVEGUIDESYSTEM,” filed Dec. 15, 2017 under Attorney Docket No. R0708.70021US01which is hereby incorporated by reference herein in its entirety.Grating coupler 1-216 may couple excitation light to waveguide 1-220,which may be configured to propagate excitation light to the proximityof one or more sample wells 1-108. Alternatively, coupling region 1-201may comprise other well-known structures for coupling light into awaveguide.

Components located off of the integrated device may be used to positionand align the excitation source 1-106 to the integrated device. Suchcomponents may include optical components including lenses, mirrors,prisms, windows, apertures, attenuators, and/or optical fibers.Additional mechanical components may be included in the instrument toallow for control of one or more alignment components. Such mechanicalcomponents may include actuators, stepper motors, and/or knobs. Examplesof suitable excitation sources and alignment mechanisms are described inU.S. patent application Ser. No. 15/161,088 titled “PULSED LASER ANDSYSTEM,” filed May 20, 2016 under Attorney Docket Number R0708.70010US02which is hereby incorporated by reference in its entirety. Anotherexample of a beam-steering module is described in U.S. patentapplication Ser. No. 15/842,720 titled “COMPACT BEAM SHAPING ANDSTEERING ASSEMBLY,” filed Dec. 14, 2017 under Attorney Docket No.R0708.70024US01 which is hereby incorporated herein by reference.

A sample to be analyzed may be introduced into sample well 1-108 ofpixel 1-112. The sample may be a biological sample or any other suitablesample, such as a chemical sample. The sample may include multiplemolecules and the sample well may be configured to isolate a singlemolecule. In some instances, the dimensions of the sample well may actto confine a single molecule within the sample well, allowingmeasurements to be performed on the single molecule. Excitation lightmay be delivered into the sample well 1-108, so as to excite the sampleor at least one fluorescent marker attached to the sample or otherwiseassociated with the sample while it is within an illumination areawithin the sample well 1-108.

In operation, parallel analyses of samples within the sample wells arecarried out by exciting some or all of the samples within the wellsusing excitation light and detecting signals from sample emission withthe photodetectors. Emission light from a sample may be detected by acorresponding photodetector and converted to at least one electricalsignal. Information regarding various characteristics of the emissionlight (e.g., wavelength, fluorescence lifetime, intensity, pulseduration and/or any other suitable characteristic) may be collected andused for subsequent analysis, as described herein. The electricalsignals may be transmitted along conducting lines (e.g., metal layers1-240) in the circuitry of the integrated device, which may be connectedto an instrument interfaced with the integrated device. The electricalsignals may be subsequently processed and/or analyzed. Processing oranalyzing of electrical signals may occur on a suitable computing deviceeither located on or off the instrument.

FIG. 1-1B illustrates a cross-sectional view of a pixel 1-112 ofintegrated device 1-102. Pixel 1-112 includes photodetection region,which may be a pinned photodiode (PPD), and a storage region, which maybe a storage diode (SD0). During operation of pixel 1-112, excitationlight may illuminate sample well 1-108 causing incident photons,including fluorescence emissions from a sample, to flow along an opticalaxis to photodetection region PPD. When transfer gate TG0 induces afirst electrical bias at the semiconductor region between photodetectionregion PPD and storage region SD0, a transfer path may be formed in thesemiconductor region. Charge carriers (e.g., photo-electrons) generatedin photodetection region PPD by the incident photons may flow along thetransfer path to storage region SD0. In some embodiments, the firstelectrical bias may be applied during a binning period during whichcharge carriers from the sample are selectively directed to storageregion SD0. Alternatively, when transfer gate TG0 provides a secondelectrical bias at the semiconductor region between photodetectionregion PPD and storage region SD0, charge carriers from photodetectionregion PPD may be blocked from reaching storage region SD0 along thetransfer path. For example, in some embodiments, drain gate REJ mayprovide a channel to drain D to draw noise charge carriers generated inphotodetection region PPD by the excitation light away fromphotodetection region PPD and storage region SD0, such as during arejection period before fluorescent emission photons from the samplereach photodetection region PPD. In some embodiments, during a readoutperiod, transfer gate TG0 may provide the second electrical bias andtransfer gate TX0 may provide an electrical bias to cause chargecarriers stored in storage region SD0 to flow to the readout region,which may be a floating diffusion (FD) region, for processing. It shouldbe appreciated that, in accordance with various embodiments, transfergates described herein may include semiconductor material(s) and/ormetal, and may include a gate of a field effect transistor (FET), a baseof a bipolar junction transistor (BJT), and/or the like.

In some embodiments, operation of pixel 1-112 may include one or morerejection (e.g., drain) periods and one or more collection periods. Inone example, operation of pixel 1-112 in accordance with one or morepulses of an excitation light source may begin with a rejection period,such as to discard charge carriers generated in pixel 1-112 (e.g., inphotodetection region PD) responsive to excitation photons from thelight source. For instance, the excitation photons may arrive at pixel1-112 prior to the arrival of fluorescence emission photons from thesample well. Transfer gates for the charge storage regions may be biasedto have low conductivity in the charge transfer channels coupling thecharge storage regions to the photodetection region, blocking transferand accumulation of charge carriers in the charge storage regions. Adrain gate for the drain region may be biased to have high conductivityin a drain channel between the photodetection region and the drainregion, facilitating draining of charge carriers from the photodetectionregion to the drain region. Transfer gates for any charge storageregions coupled to the photodetection region may be biased to have lowconductivity between the photodetection region and the charge storageregions, such that charge carriers are not transferred to or accumulatedin the charge storage regions during the rejection period.

Following the rejection period, a collection period may occur in whichcharge carriers generated responsive to the incident photons aretransferred to one or more charge storage regions. During the collectionperiod, the incident photons may include fluorescent emission photons,resulting in accumulation of fluorescent emission charge carriers in thecharge storage region(s). For instance, a transfer gate for one of thecharge storage regions may be biased to have high conductivity betweenthe photodetection region and the charge storage region, facilitatingaccumulation of charge carriers in the charge storage region. Any draingates coupled to the photodetection region may be biased to have lowconductivity between the photodetection region and the drain region suchthat charge carriers are not discarded during the collection period.Some embodiments may include multiple collection periods, such as asecond collection period following a first collection period, formultiple charge storage regions to accumulate charge carriers atdifferent times. For instance, during one of multiple collectionperiods, one of the transfer gates may be biased to facilitateaccumulation of charge carriers in the corresponding charge storageregion, and the other transfer gates may be biased to block accumulationof charge carriers in the other charge storage regions. In someembodiments, multiple charge storage regions may accumulate chargecarriers during a single collection period. In some embodiments,operation of the pixel may include as many collection periods as chargestorage regions. In some embodiments, operation as described herein maybe repeated for each pulse of the excitation light source. In someembodiments, collection periods for the various charge storage regionsmay be separated by rejection periods. For example, in some embodiments,each pulse of the excitation light source may be followed by onerejection period and one collection period (e.g., having accumulation ina single charge storage region).

As described further herein, the rejection and/or collection periods maybe controlled using one or more control signals from a control circuitof the integrated circuit, such as by providing the control signal(s) todrain and/or transfer gates of the pixel(s) of the integrated circuit.It should be appreciated that, in some embodiments, operation of pixelsdescribed herein may occur as described in this section.

For example, FIG. 1-1C is a circuit diagram of pixel 1-112 of FIG. 1-1B,according to some embodiments. In FIG. 1-1C, photodetection region PPDis coupled to multiple storage regions SD0 and SD1. Storage region SD1and transfer gate TG1 may be configured in the manner described forstorage region SD0 and transfer gate TG0. Pixel 1-112 may be configuredsuch that only one of storage regions SD0 and SD1 receive chargecarriers from photodetection region PPD at a given time. For example,storage regions SD0 and SD1 may have binning periods that are separatedin time, with transfer gate TG0 enabling flow of charge carriers tostorage region SD0 during the binning period for storage region SD0, andwith transfer gate TG1 enabling flow of charge carriers to storageregion SD1 during the binning period for storage region SD1. Likewise,pixel 1-112 may be configured such that only one of storage regions SD0and SD1 may provide charge carriers to readout region FD. For example,storage regions SD0 and SD1 may have readout periods that are separatedin time, with transfer gate TX0 enabling flow of charge carriers fromstorage region SD0 during the readout period for storage region SD0, andwith transfer gate TX1 enabling flow of charge carriers from storageregion SD1 during the readout period for storage region SD1.

FIG. 1-1D is a plan view of the pixel of FIGS. 1-1B and 1-1Cillustrating the relative positioning of storage regions SD0 and SD1relative to photodetection region PPD.

In some embodiments, some components of pixels described herein may bedisposed and/or formed on one or more substrate layers of an integratedcircuit. In some embodiments, the substrate layer(s) may alternativelyor additionally include one or more auxiliary layers (e.g., epitaxiallayers) disposed above and/or below the other substrate layer(s). Insome embodiments, some components of pixels described herein may beformed by etching away at least a portion of the substrate and/orauxiliary layer(s). In some embodiments, transfer and/or drain gatesdescribed herein may be formed using a semiconductor material such aspolysilicon, which may be at least partially opaque.

III. Techniques for Obtaining Lifetime Information

According to an aspect of the technology described herein, the inventorshave developed techniques for obtaining information regarding multiplecharacteristics of emission light from a sample well to facilitatesample analysis including sample identification with multiple dimensionsof discrimination. The inventors have recognized that fluorescentmarkers used to label biological or chemical samples, when excited byincident light, fluoresce with a characteristic lifetime (e.g., acharacteristic emission decay time period), such that analyzing thelifetime information of emission light may facilitate identification ofthe particular sample to which the fluorescent marker is attached (e.g.,bonded). Fluorescence lifetime, also referred to herein as simply“lifetime”, is a measure of the time which a fluorescent marker spendsin the excited state before returning to a ground state and emitting aphoton. In some embodiments, fluorescence lifetime information may beobtained through techniques for time binning charge carriers generatedby incident photons.

For example, FIG. 1-2A plots two different fluorescent emissionprobability curves (A and B), which can be representative of fluorescentemission from two different fluorescent molecules, for example. Withreference to curve A (dashed line), after being excited by a short orultrashort optical pulse, a probability pA(t) of a fluorescent emissionfrom a first molecule may decay with time, as depicted. In some cases,the decrease in the probability of a photon being emitted over time canbe represented by an exponential decay function p_A (t)=P_Aoe{circumflex over ( )}(−t/τ_1), where PAo is an initial emissionprobability and τ1 is a temporal parameter associated with the firstfluorescent molecule that characterizes the emission decay probability.τ1 may be referred to as the “fluorescence lifetime,” “emissionlifetime,” or “lifetime” of the first fluorescent molecule. In somecases, the value of τ1 can be altered by a local environment of thefluorescent molecule. Other fluorescent molecules can have differentemission characteristics than that shown in curve A. For example,another fluorescent molecule can have a decay profile that differs froma single exponential decay, and its lifetime can be characterized by ahalf-life value or some other metric.

A second fluorescent molecule may have a decay profile pB(t) that isexponential, but has a measurably different lifetime τ2, as depicted forcurve B in FIG. 1-2A. In the example shown, the lifetime for the secondfluorescent molecule of curve B is shorter than the lifetime for curveA, and the probability of emission pB(t) is higher sooner afterexcitation of the second molecule than for curve A. Differentfluorescent molecules can have lifetimes or half-life values rangingfrom about 0.1 ns to about 20 ns, in some embodiments.

Differences in fluorescent emission lifetimes can be used to discernbetween the presence or absence of different fluorescent moleculesand/or to discern between different environments or conditions to whicha fluorescent molecule is subjected. In some cases, discerningfluorescent molecules based on lifetime (rather than emissionwavelength, for example) can simplify aspects of an analyticalinstrument. As an example, wavelength-discriminating optics (such aswavelength filters, dedicated detectors for each wavelength, dedicatedpulsed optical sources at different wavelengths, and/or diffractiveoptics) can be reduced in number or eliminated when discerningfluorescent molecules based on lifetime. In some cases, a single pulsedoptical source operating at a single characteristic wavelength can beused to excite different fluorescent molecules that emit within a samewavelength region of the optical spectrum but have measurably differentlifetimes. An analytic system that uses a single pulsed optical source,rather than multiple sources operating at different wavelengths, toexcite and discern different fluorescent molecules emitting in a samewavelength region can be less complex to operate and maintain, morecompact, and can be manufactured at lower cost.

Although analytic systems based on fluorescent lifetime analysis canhave certain benefits, the amount of information obtained by an analyticsystem and/or detection accuracy can be increased by allowing foradditional detection techniques. For example, some analytic systems canadditionally be configured to discern one or more properties of a samplebased on fluorescent wavelength, pulse duration/width, and/orfluorescent intensity as described herein.

Referring again to FIG. 1-2A, according to some embodiments, differentfluorescent lifetimes can be distinguished with a photodetector that isconfigured to time-bin fluorescent emission events following excitationof a fluorescent molecule. The time binning can occur during a singlecharge-accumulation cycle for the photodetector. A charge-accumulationcycle is an interval between read-out events during whichphoto-generated carriers are accumulated in bins of the time-binningphotodetector. The concept of determining fluorescence lifetime bytime-binning of emission events is introduced graphically in FIG. 1-2B.At time t_(e) just prior to t₁, a fluorescent molecule or ensemble offluorescent molecules of a same type (e.g., the type corresponding tocurve B of FIG. 1-2A) is (are) excited by a short or ultrashort opticalpulse. For a large ensemble of molecules, the intensity of emission canhave a time profile similar to curve B, as depicted in FIG. 1-2B.

For a single molecule or a small number of molecules, however, theemission of fluorescent photons occurs according to the statistics ofcurve B in FIG. 1-2A, for this example. A time-binning photodetector1-322 can accumulate carriers generated from emission events intodiscrete time bins. Three bins are indicated in FIG. 1-2B, though fewerbins or more bins may be used in embodiments. The bins are temporallyresolved with respect to the excitation time t e of the fluorescentmolecule(s). For example, a first bin can accumulate carriers producedduring an interval between times t₁ and t₂, occurring after theexcitation event at time t_(e). A second bin can accumulate carriersproduced during an interval between times t₂ and t₃, and a third bin canaccumulate carriers produced during an interval between times t₃ and t₄.When a large number of emission events are summed, carriers accumulatedin the time bins can approximate the decaying intensity curve shown inFIG. 1-2B, and the binned signals can be used to distinguish betweendifferent fluorescent molecules or different environments in which afluorescent molecule is located.

Examples of a time-binning photodetector 1-322 are described in U.S.patent application Ser. No. 14/821,656, filed Aug. 7, 2015, titled“Integrated Device for Temporal Binning of Received Photons” underAttorney Docket No. R0708.70002US02 and in U.S. patent application Ser.No. 15/852,571, filed Dec. 22, 2017, titled “Integrated Photodetectorwith Direct Binning Pixel,” under Attorney Docket No. R0708.70017US01both of which are hereby incorporated herein by reference in theirentireties. For explanation purposes, a non-limiting embodiment of atime-binning photodetector is depicted in FIG. 1-2C. A singletime-binning photodetector 1-322 can comprise aphoton-absorption/carrier-generation region 1-902, a carrier-dischargechannel 1-906, and a plurality of carrier-storage regions 1-908 a, 1-908b all formed on a semiconductor substrate. Carrier-transport channels1-907 can connect between the photon-absorption/carrier-generationregion 1-902 and carrier-storage regions 1-908 a, 1-908 b. In theillustrated example, two carrier-storage regions are shown, but theremay be more or fewer. There can be a read-out channel 1-910 connected tothe carrier-storage regions. The photon-absorption/carrier-generationregion 1-902, carrier-discharge channel 1-906, carrier-storage regions1-908 a, 1-908 b, and read-out channel 1-910 can be formed by doping thesemiconductor locally and/or forming adjacent insulating regions toprovide photodetection capability, confinement, and transport ofcarriers. A time-binning photodetector 1-322 can also include aplurality of electrodes 1-920, 1-921, 1-922, 1-923, 1-924 formed on thesubstrate that are configured to generate electric fields in the devicefor transporting carriers through the device.

In operation, a portion of an excitation pulse from a pulsed opticalsource (e.g., a mode-locked laser) is delivered to a reaction chamberover the time-binning photodetector 1-322. Initially, some excitationradiation photons 1-901 may arrive at thephoton-absorption/carrier-generation region 1-902 and produce carriers(shown as light-shaded circles). There can also be some fluorescentemission photons 1-903 that arrive with the excitation radiation photons1-901 and produce corresponding carriers (shown as dark-shaded circles).Initially, the number of carriers produced by the excitation radiationcan be too large compared to the number of carriers produced by thefluorescent emission. The initial carriers produced during a timeinterval |t_(e)-t₁| can be rejected by gating them into acarrier-discharge channel 1-906 with a first transfer gate 1-920, forexample.

At a later times mostly fluorescent emission photons 1-903 arrive at thephoton-absorption/carrier-generation region 1-902 and produce carriers(indicated a dark-shaded circles) that provide useful and detectablesignal that is representative of fluorescent emission from the reactionchamber 1-330. According to some detection methods, a second electrode1-921 and third electrode 1-923 can be gated at a later time to directcarriers produced at a later time (e.g., during a second time interval|t₁-t₂|) to a first carrier-storage region 1-908 a. Subsequently, afourth electrode 1-922 and fifth electrode 1-924 can be gated at a latertime (e.g., during a third time interval |t₂-t₃|) to direct carriers toa second carrier-storage region 1-908 b. Charge accumulation cancontinue in this manner after excitation pulses for a large number ofexcitation pulses to accumulate an appreciable number of carriers andsignal level in each carrier-storage region 1-908 a, 1-908 b. At a latertime, the signal can be read out from the bins. In some implementations,the time intervals corresponding to each storage region are at thesub-nanosecond time scale, though longer time scales can be used in someembodiments (e.g., in embodiments where fluorophores have longer decaytimes).

The process of generating and time-binning carriers after an excitationevent (e.g., excitation pulse from a pulsed optical source) can occuronce after a single excitation pulse or be repeated multiple times aftermultiple excitation pulses during a single charge-accumulation cycle forthe time-binning photodetector 1-322. After charge accumulation iscomplete, carriers can be read out of the storage regions via theread-out channel 1-910. For example, an appropriate biasing sequence canbe applied to electrodes 1-923, 1-924 and at least to electrode 1-940 toremove carriers from the storage regions 1-908 a, 1-908 b. The chargeaccumulation and read-out processes can occur in a massively paralleloperation on an optoelectronic chip resulting in frames of data.

Although the described example in connection with FIG. 1-2C includesmultiple charge storage regions 1-908 a, 1-908 b in some cases a singlecharge storage region may be used instead. For example, only bin1 may bepresent in a time-binning photodetector 1-322. In such a case, a singlestorage regions 1-908 a can be operated in a variable time-gated mannerto look at different time intervals after different excitation events.For example, after pulses in a first series of excitation pulses,electrodes for the storage region 1-908 a can be gated to collectcarriers generated during a first time interval (e.g., during the secondtime interval |t₁-t₂|), and the accumulated signal can be read out aftera first predetermined number of pulses. After pulses in a subsequentseries of excitation pulses at the same reaction chamber, the sameelectrodes for the storage region 1-908 a can be gated to collectcarriers generated during a different interval (e.g., during the thirdtime interval |t₂-t₃|), and the accumulated signal can be read out aftera second predetermined number of pulses. Carriers could be collectedduring later time intervals in a similar manner if needed. In thismanner, signal levels corresponding to fluorescent emission duringdifferent time periods after arrival of an excitation pulse at areaction chamber can be produced using a single carrier-storage region.

Regardless of how charge accumulation is carried out for different timeintervals after excitation, signals that are read out can provide ahistogram of bins that are representative of the fluorescent emissiondecay characteristics, for example. An example process is illustrated inFIG. 1-2D and FIG. 1-2E, for which two charge-storage regions are usedto acquire fluorescent emission from the reaction chambers. Thehistogram's bins can indicate a number of photons detected during eachtime interval after excitation of the fluorophore(s) in a reactionchamber. In some embodiments, signals for the bins will be accumulatedfollowing a large number of excitation pulses, as depicted in FIG. 1-2D.The excitation pulses can occur at times t_(e1), t_(e2), t_(e3), . . .t_(eN) which are separated by the pulse interval time T. In some cases,there can be between 105 and 107 excitation pulses (or portions thereof)applied to a reaction chamber during an accumulation of signals in theelectron-storage regions for a single event being observed in thereaction chamber (e.g., a single nucleotide incorporation event in DNAanalysis). In some embodiments, one bin (bin0) can be configured todetect an amplitude of excitation energy delivered with each opticalpulse, and may be used as a reference signal (e.g., to normalize data).In other cases, the excitation pulse amplitude may be stable, determinedone or more times during signal acquisition, and not determined aftereach excitation pulse so that there is no bin0 signal acquisition aftereach excitation pulse. In such cases, carriers produced by an excitationpulse can be rejected and dumped from thephoton-absorption/carrier-generation region 1-902 as described above inconnection with FIG. 1-2C.

In some implementations, only a single photon may be emitted from afluorophore following an excitation event, as depicted in FIG. 1-2D.After a first excitation event at time t_(e1), the emitted photon attime to may occur within a first time interval (e.g., between times t₁and t₂), so that the resulting electron signal is accumulated in thefirst electron-storage region (contributes to bin 1). In a subsequentexcitation event at time t_(e2), the emitted photon at time t_(f2) mayoccur within a second time interval (e.g., between times t₂ and t₃), sothat the resulting electron signal contributes to bin 2. After a nextexcitation event at time t_(e3), a photon may emit at a time t_(f3)occurring within the first time interval.

In some implementations, there may not be a fluorescent photon emittedand/or detected after each excitation pulse received at a reactionchamber. In some cases, there can be as few as one fluorescent photonthat is detected at a reaction chamber for every 10,000 excitationpulses delivered to the reaction chamber. One advantage of implementinga mode-locked laser as the pulsed excitation source is that amode-locked laser can produce short optical pulses having high intensityand quick turn-off times at high pulse-repetition rates (e.g., betweenMHz and 250 MHz). With such high pulse-repetition rates, the number ofexcitation pulses within a 10 millisecond charge-accumulation intervalcan be 50,000 to 250,000, so that detectable signal can be accumulated.

After a large number of excitation events and carrier accumulations, thecarrier-storage regions of the time-binning photodetector 1-322 can beread out to provide a multi-valued signal (e.g., a histogram of two ormore values, an N-dimensional vector, etc.) for a reaction chamber. Thesignal values for each bin can depend upon the decay rate of thefluorophore. For example and referring again to FIG. 1-2B, a fluorophorehaving a decay curve B will have a higher ratio of signal in bin 1 tobin 2 than a fluorophore having a decay curve A. The values from thebins can be analyzed and compared against calibration values, and/oreach other, to determine the particular fluorophore present. For asequencing application, identifying the fluorophore can determine thenucleotide or nucleotide analog that is being incorporated into agrowing strand of DNA, for example. For other applications, identifyingthe fluorophore can determine an identity of a molecule or specimen ofinterest, which may be linked to the fluorophore.

To further aid in understanding the signal analysis, the accumulated,multi-bin values can be plotted as a histogram, as depicted in FIG. 1-2Efor example, or can be recorded as a vector or location in N-dimensionalspace. Calibration runs can be performed separately to acquirecalibration values for the multi-valued signals (e.g., calibrationhistograms) for four different fluorophores.

Thus, obtaining lifetime information obtained by the time binningtechniques described herein may facilitate analysis and identificationof a sample in a sample well. It should be appreciated that othersuitable techniques of time binning charge carriers and/or otherwiseobtaining fluorescence lifetime information other than the techniquesdescribed herein may be implemented, and aspects of the technology arenot limited in this respect

IV. Techniques for Obtaining Wavelength Information

According to another aspect of the technology described herein, theinventors developed techniques for discriminating spectral information(e.g., wavelength information) of incident light. For instance, inaddition to or alternative to using time-gating techniques fordiscriminating timing information (e.g., lifetime information), devicesdescribed herein may be configured to determine spectral information toenhance the data that can be obtained from a sample. The inventors haverecognized that, similar to lifetime, emission light from a particularfluorescent marker may have a characteristic wavelength such thatanalyzing wavelength information of emission light may facilitateidentification of the sample to which the fluorescent marker isattached. Thus, in some embodiments, emission light wavelength is anadditional dimension of discrimination used in the multi-dimensionaldiscrimination techniques described herein.

The inventors have recognized that an integrated device, such asintegrated device 1-102 may obtain wavelength measurements of emissionlight through a variety of techniques described further herein. Forexample, in some embodiments, wavelength measurements may be obtainedthrough techniques incorporating photodetector regions having differentdepths. In other embodiments, wavelength measurements may be obtainedusing techniques incorporating charge transfer channels of differentdepths. In some embodiments, wavelength information may be obtainedusing techniques incorporating optical shielding elements. Further, insome embodiments, wavelength measurements of emission light may beobtained using one or more optical sorting elements. It should beappreciated that any suitable technique in addition to or alternative tothe techniques described herein may be used to obtain wavelengthmeasurements of emission light to facilitate multi-dimensionaldiscrimination of samples, and aspects of the technology are not limitedin this respect.

a. Techniques Incorporating Regions of Different Depths

In some aspects, spectral information may be obtained using pixelshaving regions of differing depth (e.g., semiconductor junction depth).For example, in some embodiments, one or more charge storage regions areconfigured having different depths. In some embodiments, one or morephotodetection regions may be configured having different depths inaddition to or in the alternative to configuring one or more chargestorage regions with different depths. FIG. 2-1 is a side view of analternative embodiment of a pixel 2-112, which may have a time-gatedcharge storage region and a direct-excitation charge storage regionconfigured to receive incident photons from a light source via aphotodetection region, according to some embodiments. Pixel 2-112 may beconfigured in the manner described for pixel 1-112 in connection withFIGS. 1-1A to 1-1D. For instance, pixel 2-112 is shown in FIG. 2-1including photodetection region PPD and charge storage regions SD0 andSD1. In some embodiments, pixel 2-112 may also include one or moretransfer gates, such as transfer gate TG0 illustrated in FIG. 2-1 . Insome embodiments, pixel 2-112 may include one or more barriers, such asmetal layer MO illustrated in FIG. 2-1 . It should be appreciated thatpixel 2-112 may include any number of photodetection regions, chargestorage regions, and/or transfer gates. Pixel 2-112 may alternatively oradditionally include one or more drain regions and/or drain gates.

In some embodiments, pixel 2-112 may include one or more lightly p-dopedsubstrate layers. Photodetection region PPD and charge storage regionsSD0 and SD1 may be formed and/or disposed in or on the substratelayer(s) using n-type doping techniques. One or more barriers may beformed and/or disposed in or on the substrate layer(s) using p-typedoping techniques. Transfer gate TG0 may be formed using a more opaquematerial than the substrate layer(s), such as polysilicon. It should beappreciated that, in some embodiments, the substrate layer(s) may belightly n-doped, and the photodetection region PPD and charge storageregions SD0 and SD1 may be formed and/or disposed using p-type dopingtechniques. In such embodiments, the barrier(s) may be formed and/ordisposed using p-type doping techniques.

In some embodiments, some regions of pixel 2-112 may have greater depththan other regions of the pixel. For instance, in the example of FIG.2-1 , charge storage region SD1 has a greater depth than charge storageregion SD0 in a direction parallel to the optical axis (e.g., alongwhich incident light travels from the light source to pixel 2-112). Alsoshown in FIG. 2-1 , charge storage region SD1 is disposed at leastpartially below photodetection region PPD, such as incident photonsreceived from the light source and/or sample well may reach chargestorage region SD1 after passing at least partially throughphotodetection region PPD. In some embodiments, the different depths ofvarious regions of pixel 2-112 may facilitate discrimination of spectraland/or timing (e.g., lifetime) information in pixel 2-112. For instance,as shown in FIG. 2-1 , incident photons from a light source and/orsample well may travel different distances into pixel 2-112 along theoptical axis. Some photons (e.g., of a first wavelength) may reachphotodetection region PPD (as indicated by the shorter arrows in FIG.2-1 ), generating charge carriers that may be conducted to chargestorage region SD0 during a collection period. For example, during thecollection period, transfer gate TG0 may receive a control signal from acontrol circuit of pixel 2-112 responsive to which a charge transferchannel coupling photodetection region PPD to storage region SD0 maybecome more conductive. As a result, during the collection period,charge carriers generated in photodetection region PPD may travel to andaccumulate in charge storage region SD0. Other incident photons (e.g.,of a second wavelength longer than the first wavelength) may continuetraveling beyond photodetection region PPD (as indicated by the longerarrows in FIG. 2-1 ) and reach charge storage region SD1, generatingcharge carriers in charge storage region SD1. In some embodiments, theconfiguration (e.g., timing) of draining and/or collecting chargecarriers in pixel 2-112 may occur as described in connection with FIGS.1-1A to 1-1D.

In some embodiments, differences between the charge carriers collectedin charge storage region SD0 and the charge carriers collected in chargestorage region SD1 may indicate spectral and/or timing (e.g., lifetime)information of the incident light. For instance, in some embodiments, asum and/or difference of the number of charge carriers collected in thecharge storage regions may indicate a fluorescence lifetime and/orwavelength of the fluorescence emissions received from the sample well.In one example, higher wavelength photons may contribute moresubstantially to the number of charge carriers collected in chargestorage region SD0, and lower wavelength photons may contribute moresubstantially to the number of charge carriers collected in chargestorage region SD1. In this example, lower wavelength photons may havehigher energy than higher wavelength photons, causing many of the lowerwavelength photons to continue traveling beyond photodetection regionPPD to charge storage region SD1, whereas higher wavelength photons maypredominantly terminate at photodetection region PPD (e.g., due toattenuation in the bulk of pixel 2-112). As a result, the higherwavelength photons may generate more charge carriers to be collected incharge storage region SD0 during the collection period, and lowerwavelength photons may generate more charge carriers in charge storageregion SD1. Accordingly, a sum and/or difference of charge carriersaccumulated in charge storage regions SD0 and SD1 may indicate spectralinformation of the incident light, such as a wavelength of the incidentlight. In some embodiments, a depth of charge storage region SD0 and/ora depth of charge storage region SD1 may be configured such that eachcharge storage region predominantly collects incident photons having aparticular wavelength and/or range of wavelengths. Alternatively oradditionally, in some embodiments, the difference in depth betweencharge storage region(s) SD0 and/or SD1 and photodetection region PPDmay be configured such that each charge storage region predominantlycollects incident photons having a particular wavelength and/or range ofwavelengths.

In some embodiments, one or more processors (e.g., microprocessors,field programmable gate arrays (FPGAs), and/or application specificintegrated circuits (ASICs), part or each of which may be integratedwith the integrated device, etc.) coupled to pixel 2-112 may beconfigured to determine lifetime and/or spectral information based onthe number of charge carriers accumulated in charge storage region(s)SD0 and/or SD1. It should be appreciated that, alternatively oradditionally, the number of charge carriers accumulated in chargestorage region SD0 and/or SD1 may indicate a fluorescence lifetime ofthe incident light. In some embodiments, charge carriers collected inone of the charge storage regions may indicate timing information, andcharge carriers collected in another of the charge storage regions mayindicate spectral information.

It should be appreciated that integrated circuits described herein maybe configured to discriminate among incident photons having variousoptical wavelengths and/or ranges of optical wavelengths. In someembodiments, the higher wavelength photons of the above example may havea wavelength greater than 600 nm, and the lower wavelength photons mayhave a wavelength less than 600 nm. In some embodiments, the higherwavelength photons of the above example may have a wavelength greaterthan 700 nm, and the lower wavelength photons may have a wavelength lessthan 600 nm. In some embodiments, the higher wavelength photons of theabove example may have a wavelength greater than 700 nm, and the lowerwavelength photons may have a wavelength less than 700 nm. In someembodiments, the higher wavelength photons of the above example may havea wavelength greater than 600 nm, and the lower wavelength photons mayhave a wavelength less than 600 nm. In some embodiments, the higherwavelength photons of the above example may have a wavelength greaterthan 600 nm, and the lower wavelength photons may have a wavelength lessthan 550 nm. In some embodiments, the higher wavelength photons of theabove example may have a wavelength greater than 550 nm, and the lowerwavelength photons may have a wavelength less than 550 nm. In someembodiments, pixels described herein may have an area less than or equalto 40 square microns.

FIG. 2-2 is a side view of pixel 2-212, which may have a time-gatedcharge storage region and a direct-excitation charge storage regionconfigured to receive incident photons from a light source via aphotodetection region, according to some embodiments. Pixel 2-212 may beconfigured in the manner described for pixel 2-112. For instance, asshown in FIG. 2-2 , pixel 2-212 includes photodetection region PPD,charge storage regions SD0 and SD1, and transfer gate TG0. FIG. 2-2 mayalternatively or additionally include one or more barriers such as metallayer MO, and/or the barrier illustrated extending from metal layer MOinto the pixel. For instance, the barrier may be elongated in adirection parallel to the optical axis along which photodetection regionPPD receives incident light from a light source. While pixel 2-112 ofFIG. 2-1 may be configured to receive light in the direction in whichphotodetection region PPD is spaced from transfer gate TG0 parallel tothe optical axis, pixel 2-212 may be configured to receive light in anopposite direction from pixel 2-112. For instance, as shown in FIG. 2-2, pixel 2-112 may be configured to receive light in a direction in whichtransfer gate TG0 is spaced from photodetection region PPD parallel tothe optical axis. One advantage of the structure illustrated in FIG. 2-2(e.g., back-side illumination) is that incident photons may beattenuated in the substrate layer(s) of the pixel prior to reaching thedepth of the charge storage regions, thus reducing the number of noisephotons and/or charge carriers that may reach and/or be generated in thecharge storage regions, thus improving signal integrity of pixeloperation.

As in FIG. 2-1 , some regions of pixel 2-212 may have greater depth thanother regions of the pixel. For instance, in the example of FIG. 2-2 ,charge storage region SD1 has a greater depth than charge storage regionSD0 in a direction parallel to the optical axis (e.g., along whichincident light travels from the light source to pixel 2-112).Alternatively or additionally, one of charge storage regions SD0 and SD1may be configured to receive incident light and generate and storecharge carriers. For instance, in FIG. 2-2 , charge storage region SD1is positioned such that incident photons may travel throughphotodetection region PPD and reach charge storage region SD0. In someembodiments, the barrier(s) of pixel 2-212 may block at least some ofthe incident photons (e.g., obliquely incident photons) from reachingcharge storage region SD0, such that charge carriers accumulated incharge storage region SD0 are predominantly time-gated fromphotodetection region PPD using transfer gate TG0. In some embodiments,the difference in depth between charge storage regions SD0 and SD1,and/or photodetection region PPD, may cause charge carriers accumulatedin the charge storage regions to have different indicate timing and/orspectral information of the incident light. For instance, in someembodiments, a depth of charge storage region SD0, a depth of chargestorage region SD1, and/or a depth of photodetection region PPD may beconfigured such that each charge storage region predominantly collectsincident photons having a particular wavelength and/or range ofwavelengths.

FIG. 2-3 is a side view of pixel 2-312, which may have a time-gatedcharge storage region, a direct-excitation charge storage regionconfigured to receive incident photons from a light source, and aphotodetection region configured to receive at least some of theincident photons via the direct-excitation charge storage region,according to some embodiments. Pixel 2-312 may be configured in themanner described for pixel 2-312 in connection with FIG. 2-3 . Forinstance, in FIG. 2-3 , pixel 2-312 includes photodetection region PPD,charge storage regions SD0 and SD1, and transfer gate TG0. In someembodiments, pixel 2-312 may include one or more barriers such as metallayer MO and/or the barrier shown elongated parallel to the opticalaxis. Also shown in FIG. 2-3 , pixel 2-312 may be configured to receivelight in the direction in which transfer gate TG0 is spaced fromphotodetection region PPD parallel to the optical axis.

In some embodiments, the difference in depth between charge storageregions SD0 and SD1, and/or photodetection region PPD, may cause chargecarriers accumulated in the charge storage regions to have differentindicate timing and/or spectral information of the incident light. Forinstance, in some embodiments, a depth of charge storage region SD0, adepth of charge storage region SD1, and/or a depth of photodetectionregion PPD may be configured such that each charge storage regionpredominantly collects incident photons having a particular wavelengthand/or range of wavelengths. However, unlike in FIG. 2-2 ,photodetection region PPD of pixel 2-312 is configured to receiveincident photons having traveled through charge storage region SD0. As aresult, a greater number of lower wavelength photons may reachphotodetection region PPD than higher wavelength photons, such that agreater number of lower wavelength charge carriers may be generated andstored in charge storage region SD1 than higher wavelength chargecarriers. Likewise, a greater number of higher wavelength photons maygenerate high wavelength charge carriers in photodetection region PPD,resulting in a greater number of higher wavelength charge carriersaccumulated in charge storage region SD0 by time-gating via transfergate TG0.

FIG. 2-4 is a side view of pixel 2-412 having two photodetection regionsof different depths and two time-gated charge storage regions, accordingto some embodiments. Pixel 2-412 may be configured in the mannerdescribed for pixel 2-112 in connection with FIG. 2-1 . For instance, asshown in FIG. 2-4 , pixel 2-412 includes photodetection region PPD0,charge storage regions SD0 and SD1, and transfer gate TG0. Like in FIG.2-1 , pixel 2-412 may include regions of different depths. For instance,in FIG. 2-4 , pixel 2-412 may include photodetection region PPD1 andtransfer gate TG1, and photodetection regions PPD0 and PPD1 are shownhaving different depths. In one example, a greater number of lowerwavelength incident photons may reach photodetection region PPD1 thanhigher wavelength incident photons. As a result, a greater number oflower wavelength charge carriers may be accumulated in charge storageregion SD1 by time-gating via transfer gate TG1 than higher wavelengthcharge carriers. Likewise, a greater number of higher wavelengthincident photons may generate charge carriers in photodetection regionPPD0 than lower wavelength incident photons, resulting in more higherwavelength charge carriers accumulated in charge storage region SD0 bytime-gating via transfer gate TG0 than lower wavelength charge carriers.

FIG. 2-5 is a side view of pixel 2-512 having three photodetectionregions of different depths, according to some embodiments. Pixel 2-512may be configured in the manner described for pixel 2-415 in connectionwith FIG. 2-4 , such as having regions of different depths. Forinstance, as shown in FIG. 2-5 , pixel 2-512 includes photodetectionregions PPD0 and PPD1 and charge storage regions SD0 and SD1, withphotodetection regions PPD0 and PPD1 having different depths. Also shownin FIG. 2-5 , pixel 2-512 includes photodetection region PPD2 and chargestorage region SD2, with photodetection region PPD2 having a differentdepth from photodetection regions PPD0 and PPD1. In some embodiments,such as illustrated in FIG. 2-5 , one or more charge storage regions maybe positioned between adjacent ones of the photodetection regions.Alternatively or additionally, as illustrated in FIG. 2-5 , pixel 2-512may include one or more barriers 2-502 (e.g., p-doped barriers “Pwell”)positioned between adjacent photodetection regions PD. For example,barriers 2-502 may be doped with an opposite conductivity type thanphotodetection regions PPD0, PPD1, PPD2, such as being p-type doped whenphotodetection regions PPD0, PPD1, and PPD2 are n-type doped.

It should be appreciated that, in embodiments having more than twophotodetection regions, some (or all) of the photodetection regions mayhave a same depth. Alternatively or additionally, in some embodimentshaving multiple photodetection regions (e.g., having different depths),the charge storage regions may have different depths. By including moreregions of different depths, such as illustrated in FIG. 2-5 , moretiming and/or spectral information of the incident light may be obtainedfor processing.

b. Techniques Incorporating Charge Transfer Channels of Different Depths

FIGS. 3-1A and 3-1B illustrate pixel 3-112, which may have onephotodetection region and four charge storage regions, according to someembodiments. FIG. 3-1A is a top view of pixel 3-112, and FIG. 3-1B is across-sectional view of pixel 3-112 along line A. Pixel 3-112 may beconfigured in the manner described for pixel 2-412 in connection withFIG. 2-4 . For instance, as shown in FIGS. 3-1A and 3-1B, pixel 3-112includes photodetection regions PPD0 and PPD1, as well as charge storageregions SD0 and SD1 (of which charge storage region SD1 is hidden fromview), and transfer gates TG0 and TG1. In addition, pixel 3-112 includescharge storage regions SD2 and SD3 (of which charge storage region SD2hidden from view) and transfer gates TG2 and TG3 positioned on anopposite side of photodetection regions PPD0 and PPD1 from chargestorage regions SD0 and SD1 and transfer gates TG0 and TG1. In oneexample, charge storage regions SD0 and SD1 may be configuredsubstantially identically to charge storage regions SD2 and SD3,respectively. For instance, charge storage regions SD0 and SD1 may havea same depth and may be configured to receive charge carriers fromphotodetection region PPD0 via charge transfer channels having a samedepth as well. Likewise, charge storage regions SD2 and SD3 may have asame depth and may be configured to receive charge carriers fromphotodetection region PPD1 via charge transfer channels having a samedepth. In some embodiments, charge storage regions SD0 and SD3 may beconfigured substantially identically to one another, and charge storageregions SD1 and SD2 may be configured substantially identically to oneanother. In some embodiments, charge storage regions SD0 and SD2 may beconfigured substantially identically to one another, and charge storageregions SD1 and SD3 may be configured substantially identically to oneanother. It should be appreciated that slight differences, such as dueto inconsistencies in manufacturing pixel 3-112, may result in greaterdifferences between substantially identically configured charge storageregions. In some embodiments, charge storage regions of pixel 3-112 maybe configured differently from one another, such has having differentdepths from one another.

Regions of pixel 3-112 may have different depths. In some embodiments,charge storage regions SD0 and SD3 may have a same depth, and chargetransfer channels coupling respective photodetection regions PPD0 andPPD1 to charge storage regions SD0 and SD3 may have different depths,such as shown in FIG. 3-1B. For instance, as shown in FIG. 3-112 , thecharge transfer channel coupled to charge storage region SD3 is at leastpartially below the charge transfer channel coupled to charge storageregion SD0 (e.g., in a direction parallel to the optical axis).Accordingly, in some embodiments, charge storage regions SD0 and SD1 mayaccumulate a substantially equal amount of charge carriers over a samerange of frequencies (e.g., a same number of lower wavelength chargecarriers and a same number of higher wavelength charge carriers).Likewise, in some embodiments, charge storage regions SD2 and SD3 mayaccumulate a substantially equal amount of charge carriers over therange of frequencies as one another, but different numbers of chargecarriers at the various frequencies than charge storage regions SD0 andSD1. For example, by receiving charge carriers from deeperphotodetection region PPD1, charge storage regions SD2 and SD3 mayaccumulate a greater number of higher charge carriers than chargestorage regions SD0 and SD1. Accordingly, charge carriers accumulated inthe charge storage regions may indicate spectral information of theincident light. Moreover, including multiple charge storage regions SD0and SD1 (and likewise SD2 and SD3), the charge carriers may be read outto determine both timing and spectral information. For instance, in oneexample, charge carriers accumulated in charge storage regions SD0 andSD3 may indicate spectral information, and charge carriers accumulatedin charge storage regions SD0 and SD1 may indicate timing information.It should be appreciated that other combinations of charge storageregions SD0, SD1, SD2, and SD3 may be used to determine variousinformation of the incident light.

FIGS. 3-2A and 3-2B illustrate pixel 3-212, which may have onephotodetection region and six charge storage regions, according to someembodiments. FIG. 3-2A is a top view of pixel 3-212, and FIG. 3-2B is across-sectional view of pixel 3-212 along line B. Pixel 3-212 may beconfigured in the manner described for pixel 3-112 in connection withFIGS. 3-1A and 3-1B. For instance, pixel 3-112 is shown in FIGS. 3-2Aand 3-2B including photodetection regions PPD0 and PPD1, transfer gatesTG0, TG1, TG2, and TG4, and charge storage regions SD0, SD1, SD2, andSD3 (of which the charge storage regions are hidden from view). Inaddition, pixel 3-212 includes photodetection region PPD2, transfergates TG4 and TG5, and charge storage regions SD4 and SD5 (of whichcharge storage region SD5 is hidden from view). As shown in FIG. 3-2B,photodetection region PPD2 may have a different depth fromphotodetection regions PPD0 and PPD1. In one example, charge storageregions SD4 and SD5 may be configured to receive charge carriers fromphotodetection region PPD2 via charge transfer channels having a depthdifferent from the charge transfer channels coupling photodetectionregions PPD0 and PPD1 to charge storage regions SD0, SD1, SD2, and SD3.Accordingly, in some embodiments, charge storage regions SD4 and SD5 mayaccumulate a greater number of lower wavelength charge carriers than theother charge storage regions, such as due to more lower wavelengthphotons reaching photodetection region PPD2 than the otherphotodetection regions. As a result, including greater numbers ofphotodetection regions and charge storage regions may increase thetiming and/or spectral information of the incident light that may beobtained using pixel 3-212.

c. Techniques Incorporating Optical Shielding

It should be appreciated that, in some embodiments, spectral informationmay be obtained using a pixel that does not incorporate regions ofdifferent depths. For instance, FIGS. 3-3A to 3-3C illustrate pixel3-312, which may have one photodetection region and two charge storageregions, according to some embodiments. FIG. 3-3A is a top view of pixel3-312, FIG. 3-3B is a cross-sectional view of Pixel 3-312 along line Aof FIG. 3-3A, and FIG. 3-3C is a cross-sectional view of pixel 3-312along line B of FIG. 3-3C. Pixel 3-312 may be configured in the mannerdescribed for pixel 2-212 in connection with FIG. 2-2 . For instance, asshown in FIGS. 3-3A to 3-3C, pixel 3-312 includes photodetection regionPD, charge storage regions SD0 and SD1, and transfer gate TG0. Alsoshown in FIGS. 3-3B and 3-3C, pixel 3-312 may be configured to receiveincident light in the direction in which transfer gate TG0 is spacedfrom photodetection region PPD parallel to the optical axis. Pixel 3-312may also include transfer gate TG1. For instance, one or more barriers(e.g., metal layer MO) may be configured to block incident photons fromreaching charge storage region SD0, such that charge carrierspredominantly reach charge storage region SD0 by time-gating viatransfer gate TG0 from photodetection region PD. In some embodiments,one or more barriers may be positioned adjacent charge storage regionSD0 and/or between the photodetection region PPD and charge storageregion SD0, elongated parallel to the optical axis, such as illustratedin FIG. 3-3C. In contrast to charge storage region SD0, charge carriersmay reach charge storage region SD1 by direct excitation (e.g., byincident photons reaching charge storage region SD1 and generatingcharge carriers), in addition to time-gating via transfer gate TG1 fromphotodetection region PD. Accordingly, charge carriers accumulated inthe respective charge storage regions SD0 and SD1 may indicate timingand/or spectral information. In one example, a difference in the numberof charge carriers accumulated in the respective charge storage regionsmay indicate a difference in the wavelength content of the incidentphotons (e.g., a quantity of the incident light that is lowerwavelength, a quantity that is higher wavelength, etc.).

FIG. 3-4 is another example of a pixel 3-412, the pixel 3-412 of FIG.3-4 having at least one charge storage region which is opticallyshielded, for example, by a barrier. For example, pixel 3-412 may beconfigured having one photodetection region PD, and two charge storageregions SNA and SNB. Each of the charge storage regions may be coupledto readout regions FDA and FDB, respectively, for causing chargecarriers to flow out of the charge storage regions SNA and SNB and intoreadout regions FDA and FDB for processing. In some embodiments, readoutregions FDA and FDB are floating diffusion regions. Emission lightincident on photodetection region PPD may generate charge carriers whichare selectively stored in one of the two charge storage regions SNA andSNB. Pixel 3-312 may be configured to operate in a “ping pong” modealternating, at different time intervals, between reading out chargecarriers in each of charge storage regions SNA and SNB, as is describedherein, for example, with respect to FIG. 4-4 .

As shown in FIG. 3-4 , at least one of the charge storage regions may beoptically shielded, for example, using a barrier, to restrict opticalpaths to the charge storage region other than through photodetectionregion PD. For example, in FIG. 3-4 , a barrier is configured such thatincident photons do not reach charge storage region SNA. Instead, chargestorage region SNA may receive charge carriers through photodetectionregion PPD according to the methods described herein. A second chargestorage region, SNB, may not be optically shielded and thus mayaccumulate charge carriers from photodetection region PPD as well asgenerating charge carriers directly from incident emission light whichhave longer wavelengths and thus can reach charge storage region SNB.Thus, analyzing the differences in charge accumulation between chargestorage regions SNA and SNB may effectively provide wavelengthinformation of incident emission light.

d. Techniques Incorporating One or More Optical Sorting Elements

FIG. 4-1 is a side view of pixel 4-112, which may have an opticalsorting element, two photodetection regions, and two time-gated chargestorage regions, according to some embodiments. Pixel 4-112 may beconfigured in the manner described for pixel 2-412 in connection withFIG. 2-4 . For instance, pixel 4-112 is shown in FIG. 4-1 includingphotodetection regions PPD0 and PPD1, charge storage regions SD0 andSD1, and transfer gates TG0 and TG1. Photodetection regions PPD0 andPPD1 are shown having substantially equal depths, as are charge storageregions SD0 and SD1. Pixel 4-112 is shown further including opticalsorting element OSE configured to direct at least some incident photonsfrom the light source towards charge storage region SD0 and at leastsome other incident photons from the light source towards charge storageregion SD1. The charge carriers accumulated in charge storage regionsSD0 and SD1 responsive to the incident photons may be indicative ofdifferent information. For instance, charge storage region SD0 mayindicate timing and/or spectral information particular to the incidentphotons directed towards charge storage region SD0, and charge storageregion SD1 may indicate such information for the incident photonsdirected towards charge storage region SD1. In some embodiments, thecharge carriers stored in charge storage region SD0 may indicate timinginformation and the charge carriers stored in charge storage region SD0may indicate wavelength information. Alternatively or additionally,information from each charge storage region may be combined to determinetiming and/or spectral information of the incident light received byeach charge storage region.

In accordance with various embodiments, optical sorting element OSE maybe configured as at least partially refractive, diffractive, scattering,and/or plasmonic. For instance, in some embodiments, the optical sortingelement OSE may include a micro-disk, a micro-lens, and/or a prismconfigured to refract incident light towards the charge storage regionsSD0 and SD1, such as depending on a wavelength of the incident light. Insome embodiments, the optical sorting element OSE may include a lineargrating element, a curved grating element, a zone plate, and/or aphotonic crystal configured to diffract the incident light towards thecharge storage regions SD0 and SD1, such as depending on the wavelengthof the incident light. In some embodiments, the optical sorting elementOSE may include a scattering element, such as having multiple elementswith different refractive indices. In some embodiments, the opticalsorting element OSE may include a plasmonic element, such as nano-holesand/or an extraordinary optical transmission element. Because theoptical sorting element OSE may cause incident photons having differentfrequencies towards the different photodetection regions PPD0 and PPD1,charge carriers accumulated in the charge storage regions SD0 and SD1may be indicative of different spectral information of the incidentphotons, such as different wavelength information.

It should be appreciated that, in some embodiments, one or more of thecharge storage regions may be configured to receive incident photonsfrom the optical sorting element OSE and generate and store chargecarriers in response.

FIG. 4-2 is a side view of pixel 4-212, which may have an opticalsorting element, two photodetection regions, and two time-gated chargestorage regions, according to some embodiments. Pixel 4-212 may beconfigured in the manner described for pixel 4-112 in connection withFIG. 4-1 . For instance, pixel 4-212 is shown in FIG. 4-2 havingphotodetection regions PPD0 and PPD1, charge storage regions SD0 andSD1, and transfer gates TG0 and TG1. In addition, pixel 4-212 includesone or more barriers positioned at least partially betweenphotodetection regions PPD0 and PPD1 and respective charge storageregions SD0 and SD1. Also shown in FIG. 4-2 , pixel 4-212 may beconfigured to receive incident photons in a direction in which transfergates TG0 and TG1 are spaced from photodetection regions PPD0 and PPD1parallel to the optical axis, whereas pixel 4-112 may be configured toreceive incident photons in a direction in which photodetection regionsPPD0 and PPD1 are spaced from transfer gates TG0 and TG1 parallel to theoptical axis.

V. Techniques for Obtaining Pulse Duration Information

According to a further aspect of the technology described herein, theinventors have developed techniques for obtaining pulse durationinformation for emission light from a sample in a sample well. Forexample, fluorescent markers bound to molecules in a sample may havecharacteristic pulse and interpulse durations such that obtainingmeasurements of pulse and interpulse duration of emission light from aparticular fluorescent marker facilitates identifying the fluorescentmarker from which light is emitted. Pulse duration, also referred toherein as pulse width, refers to the interval of time measured across apulse, in some embodiments, at the full width half maximum of a pulse.Interpulse duration, also referred to herein as interpulse width, refersto the interval of time between pulses.

Thus, in some embodiments, the integrated device described herein may beconfigured to implement techniques for obtaining pulse durationinformation, such as the techniques described in U.S. patent applicationSer. No. 16/686,028 titled “METHODS AND COMPOSITIONS FOR PROTEINSEQUENCING,” filed Nov. 15, 2019 under Attorney Docket No.R0708.70042US02 and PCT Application No. PCT/US19/61831 titled “METHODSAND COMPOSITIONS FOR PROTEIN SEQUENCING,” filed Nov. 15, 2019 underAttorney Docket No. R0708.70042WO00, both which are incorporated byreference in their entireties. For example, as described herein,molecules in a sample may be labeled with fluorescent markers. One ormore fluorescent markers may attach (e.g., bond) to a molecule, and,upon being excited by excitation light, may each emit a photon,collectively referred to as a fluorescence event. Emitted photonsgenerated by many such fluorescent events due to repeated excitation ofthe fluorescent markers may be referred to as a signal pulse. Eachsignal pulse comprises a pulse duration (“pd”) corresponding to anassociation event between a recognition molecule of the fluorescentmarkers and the sample molecule under analysis.

For example, without wishing to be bound by theory, labeled affinityreagent of a fluorescent marker selectively binds with a recognitionmolecule according to a binding affinity (K_(D)) defined by anassociation rate, or an “on” rate, of binding (k_(on)) and adissociation rate, or an “off” rate, of binding (k_(off)). When thefluorescent molecule is bound to the recognition molecule, the markermay fluoresce and emit a photon, while, when the fluorescent marker isunbound, the marker may not fluoresce even when receiving excitationlight and entering and exiting an excited state, as described herein.The rate constants k_(off) and k_(on) are the critical determinants ofpulse duration (e.g., the time corresponding to a detectable bindingevent) and interpulse duration (e.g., the time between detectablebinding events), respectively. In some embodiments, these rates can beengineered to achieve pulse durations and pulse rates (e.g., thefrequency of signal pulses) that give the best sequencing accuracy.

Thus, in some embodiments, the pulse duration is characteristic of adissociation rate of binding. In addition, each signal pulse of acharacteristic pattern is separated from another signal pulse of thecharacteristic pattern by an interpulse duration (“ipd”). In someembodiments, the interpulse duration is characteristic of an associationrate of binding. In some embodiments, a change in magnitude (“ΔM”) canbe determined for a signal pulse based on a difference between baselineand the peak of a signal pulse. In some embodiments, a characteristicpattern is determined based on pulse duration. In some embodiments, acharacteristic pattern is determined based on pulse duration andinterpulse duration. In some embodiments, a characteristic pattern isdetermined based on any one or more of pulse duration, interpulseduration, and change in magnitude. In some embodiments, the series ofpulses provide a pulsing pattern (e.g., a characteristic pattern) whichmay be diagnostic of the identity of the sample under analysis.

FIGS. 4-3A-C show non-limiting examples of characteristic pulse patternsfor sample molecules under analysis. FIG. 4-3A is a plot showingdiscriminant pulse durations (time duration of signal peaks) among thethree N-terminal residues when reversibly bound by labeled ClpS2. FIG.4-3B is a plot showing discriminant interpulse durations (time durationbetween signal pulses) among the three N-terminal residues. FIG. 4-3C-Eshows plots which further illustrate the discriminant pulse durationsamong phenylalanine, tyrosine, and tryptophan at peptide N-termini.

As described herein, signal pulse information may be used to identify amolecule, such as an amino acid, based on a characteristic pattern in aseries of signal pulses. In some embodiments, a characteristic patterncomprises a plurality of signal pulses, each signal pulse comprising apulse duration. In some embodiments, the plurality of signal pulses maybe characterized by a summary statistic (e.g., mean, median, time decayconstant) of the distribution of pulse durations in a characteristicpattern. In some embodiments, the mean pulse duration of acharacteristic pattern is between about 1 millisecond and about 10seconds (e.g., between about 1 ms and about 1 s, between about 1 ms andabout 100 ms, between about 1 ms and about 10 ms, between about 10 msand about 10 s, between about 100 ms and about 10 s, between about 1 sand about 10 s, between about 10 ms and about 100 ms, or between about100 ms and about 500 ms). In some embodiments, different characteristicpatterns corresponding to different types of amino acids in a singlepolypeptide may be distinguished from one another based on astatistically significant difference in the summary statistic. Forexample, in some embodiments, one characteristic pattern may bedistinguishable from another characteristic pattern based on adifference in mean pulse duration of at least 10 milliseconds (e.g.,between about 10 ms and about 10 s, between about 10 ms and about 1 s,between about 10 ms and about 100 ms, between about 100 ms and about 10s, between about 1 s and about 10 s, or between about 100 ms and about 1s). It should be appreciated that, in some embodiments, smallerdifferences in mean pulse duration between different characteristicpatterns may require a greater number of pulse durations within eachcharacteristic pattern to distinguish one from another with statisticalconfidence.

As described herein, fluorescence markers may be configured such thatthe fluorescence markers only fluoresce when attached to a molecule ofinterest in the sample, and may not fluoresce during periods ofdisassociation. For example, in some embodiments, a fluorescenceresonance energy transfer (FRET) technique is used such thatfluorescence of markers selectively occur when attached to a molecule ofinterest in the sample. Labeled affinity reagent for a fluorescentmarker may comprise a label having binding-induced luminescence. Forexample, in some embodiments, a labeled aptamer comprises a donor labeland an acceptor label. Labeled aptamer as a free molecule may adopt aconformation in which donor label and acceptor label are separated by adistance that limits detectable FRET between the labels (e.g., about 10nm or more). Labeled aptamer as a selectively bound molecule adopts aconformation in which donor label and acceptor label are within adistance that promotes detectable FRET between the labels (e.g., about10 nm or less). In yet other embodiments, labeled aptamer comprises aquenching moiety and functions analogously to a molecular beacon,wherein luminescence of labeled aptamer is internally quenched as a freemolecule and restored as a selectively bound molecule (see, e.g.,Hamaguchi, et al. (2001) Analytical Biochemistry 294, 126-131). Withoutwishing to be bound by theory, it is thought that these and other typesof mechanisms for binding-induced luminescence may advantageously reduceor eliminate background luminescence to increase overall sensitivity andaccuracy of the methods described herein.

The inventors have recognized that obtaining one or more measurementsfor pulse duration and/or interpulse duration to facilitate thediscrimination techniques described herein may be accomplished using theintegrated device 1-102 and the time binning techniques describedherein, particularly with respect to Section III of this disclosure. Insome embodiments, pulse duration and/or interpulse duration may be usedas dimensions for discriminating a sample in a sample well underanalysis. For example, as described herein, particular molecules mayhave a characteristic pulse duration and/or interpulse duration and asample may be identified by comparing measured pulse durations and/orinterpulse duration with known characteristic durations.

VI. Techniques for Obtaining Intensity Information

According to another aspect of the technology described herein, theinventors have developed techniques for obtaining one or moremeasurements of emission light intensity which can, in some embodiments,facilitate multi-dimensional discrimination techniques of a sample in asample well. The inventors have recognized that some fluorophores mayemit at significantly different intensities or have a significantdifference in their probabilities of excitation (e.g., at least adifference of about 35%) even though their decay rates may be similar.By referencing binned signals (e.g., by measuring the accumulation ofcharge carriers) to measured excitation energy and/or other acquiredsignals, it can be possible to distinguish different fluorophores basedon intensity levels. Thus, it should be appreciated that an integrateddevice, such as integrated device 1-102, may be configured to measureintensity based on the accumulation of charge carriers in a storage binthrough various read-out periods as described herein, and measurementsof intensity may be used to distinguish the particular sample underanalysis.

In some embodiments, different numbers of fluorophores of the same typemay be linked to different molecules in a sample, so that each moleculemay be identified based on luminescence intensity. For example, twofluorophores may be linked to a first labeled molecule and four or morefluorophores may be linked to a second labeled molecule. Because of thedifferent numbers of fluorophores, there may be different excitation andfluorophore emission probabilities associated with the differentmolecules. For example, there may be more emission events for the secondlabeled molecule during a signal accumulation interval, so that theapparent intensity of the bins is significantly higher than for thefirst labeled molecule. Thus, the inventors have recognized that, insome embodiments, controlling the number of fluorophores which arelinked to a particular molecule in a sample may facilitateidentification of the sample. In some embodiments, intensity istherefore at least one of a number of characteristics used inmulti-dimensional discrimination techniques for sample analysis.

VII. Applications of Multi-dimensional Discrimination Techniques

Having thus described multiple techniques for acquiring informationregarding various characteristics (e.g., lifetime, wavelength,intensity, pulse duration, and/or interpulse duration) of emission lightfrom a sample, example applications of the multi-dimensionaldiscrimination techniques will now be described. For example, theinventors have recognized that identification of one or more moleculesin a sample under analysis may be identified using the multi-dimensionaltechniques described herein. In particular, measurements for one or morecharacteristics of emission light may be obtained by a device, such asthe integrated device described herein, and the collected measurementsmay be compared to known characteristic values of the measuredcharacteristics for a fluorescent marker to determine which fluorescentmarker is the most likely source of the emission light. In turn, byidentifying the fluorescent marker, the identity of the molecule towhich the fluorescent marker is attached can be known based on theparticular type of molecule to which the fluorescent marker is known toattach.

Any suitable combination of characteristics may be combined and used inthe multi-dimensional techniques described herein. For example, in someembodiments, a two-dimensional discrimination technique may identify asample of interest based on information of any two of lifetime,wavelength, pulse duration, interpulse duration, and intensity ofemission light associated with the sample. In some embodiments, atwo-dimensional discrimination technique for identifying a sample ofinterest is based on wavelength information and lifetime information ofemission light associated with the sample. In some embodiments, atwo-dimensional discrimination technique for identifying a sample ofinterest is based on lifetime information and intensity information ofemission light associated with the sample. In some embodiments, atwo-dimensional discrimination technique for identifying a sample ofinterest is based on wavelength information and lifetime information ofemission light associated with the sample. In some embodiments, athree-dimensional discrimination technique may identify a sample ofinterest based on information of any three of lifetime, wavelength,pulse duration, interpulse duration, and intensity of emission lightassociated with the sample. In some embodiments, a three-dimensionaldiscrimination technique for identifying a sample of interest is basedon lifetime information, wavelength information, and intensityinformation of emission light associated with the sample. In someembodiments, a three-dimensional discrimination technique foridentifying a sample of interest is based on any two of wavelengthinformation, lifetime information, and intensity information, and anyone of pulse duration information and interpulse duration information ofemission light associated with the sample. According to another aspectof the technology described herein, a four-dimensional discriminationtechnique is used to identify a sample of interest based information ofany four of lifetime, wavelength, pulse duration, interpulse duration,and intensity of emission light associated with the sample. In someembodiments, a four-dimensional discrimination technique for identifyinga sample of interest is based on lifetime information, wavelengthinformation, intensity information and one of interpulse durationinformation and pulse duration information of emission light associatedwith the sample. According to another aspect of the technology describedherein, a five-dimensional discrimination technique is used to identifya sample of interest based on information of lifetime, wavelength,intensity, pulse duration, and interpulse duration of emission lightassociated with the sample.

In some embodiments, a two-dimensional discrimination technique foridentifying a sample of interest is based on measurements of any two ofwavelength, lifetime, intensity, pulse duration and interpulse durationof emission light associated with the sample. In some embodiments, atwo-dimensional discrimination technique for identifying a sample ofinterest is based on measurements of lifetime and intensity of emissionlight associated with a sample. In some embodiments, a two-dimensionaldiscrimination technique for identifying a sample of interest is basedon measurements of wavelength and intensity of emission light associatedwith a sample.

For example, FIG. 4-4 shows one example of data collected for amulti-dimensional discrimination technique, according to someembodiments described herein. In the illustrated example in FIG. 4-4 ,measurements are collected for intensity, wavelength, and lifetime ofemission light collected emitted from a sample within a sample well andcollected by a photodetector. The graph shown in FIG. 4-4 containssignal data collected from two storage nodes, SNA and SNB. The storagenodes SNA and SNB may be configured as the two charge storage regions inFIG. 3-3 , for example. “Ping” and “pong” labels as applied to storagenodes SNA and SNB may refer to time intervals at which signal from arespective storage node is obtained by a processor. Charge carriersaccumulated in storage regions may be read out to a component, such as aprocessor, controller, and/or circuit, incrementally, with chargescarriers in a first storage node SNA being readout twice initially (aping interval, and a pong interval) and thereafter charge carriers in asecond storage node SNB being read out twice (a ping interval, and apong interval). The signal represents the accumulated charge from allstorage nodes previously read out.

Analysis of the signal at the various time intervals providesinformation for various characteristics of incident emission light. Forexample, intensity of the incident emission light may be analyzed bestby comparing relative signal measurements for the SNA ping interval,which is the initial interval of charge carrier read out. As shown inthe graph and accompanying table, pulses 1-4 have a relatively lowersignal than pulses 5-8, and thus, pulses 1-4 can be considered as havinga low intensity while pulses 5-8 can be considered as having arelatively higher intensity. Lifetime information can be analyzed byviewing the relative difference in signal between the SNA ping intervaland the SNA pong interval. For example, where the SNA pong intervalsignal read out is approximately the same as the SNA ping interval, muchof the signal was generated by charge carriers generated during the SNAping interval, which is the earlier interval, and therefore for thosepulses, lifetime is relatively short. Where the SNA pong interval signalread out is larger than the SNA ping interval, more charge carriers weregenerated at a later time interval and thus lifetime is relatively long.In the illustrated figure, odd pulses are shown to have short lifetimeswhile even pulses have longer lifetimes. Finally, wavelength informationcan be determined based on the arrangement of the storage nodes SNA andSNB. When the storage nodes are configured with an optical blockingelement as described with respect to FIG. 3-3 , the storage node whichis unblocked, SNB, will receive more light at longer wavelengths thanthe blocked storage node, SNA. Thus, analyzing the difference betweenSNA and SNB pong intervals to determine whether the SNB charge carrierreadouts are relatively larger than SNA charge carrier readouts willindicate whether the pulse has a relatively short or long wavelength.For example, comparing SNA and SNB readout signals for pong intervalsand determining whether the signal readout for SNB is approximately morethan double the SNA readout signal will determine whether the wavelengthof incident emission light is relatively short or otherwise, isrelatively long.

The inventors have recognized that discrimination techniques arelatively higher number of dimensions (e.g., three dimensions vs. twodimensions, four dimensions vs. three dimensions, etc.) may improve theability to accurately identify the source of emission light andtherefore to accurately identify the sample under analysis. As describedherein, in the application of the multidimensional discriminationtechniques to protein sequencing, in order to accurately identify 20different amino acids as well as posttranslational modifications, it isadvantageous to use discrimination techniques having higher degrees ofdimensionality (e.g., three, four, and/or five dimensions fordiscriminating a sample). In particular, higher dimensional techniquesmay facilitate accurate identification of a emission source (e.g., afluorescent marker and/or a sample to which the fluorescent marker isattached) even when the received signal is relatively low (i.e., theemission light intensity is relatively low). The inventors haverecognized that developing discrimination techniques which function evenwith relatively low signal may extend read length for the sequencingapplications described herein and improve the scalability of thetechnology to larger assay sizes.

Although it may be advantageous to increase the dimensionality of adiscrimination technique used to identify a sample, doing so mayincrease the complexity of the integrated device from which thediscrimination information is obtained. In addition, providingfluorescent markers optimized for obtaining information on multiplecharacteristics (e.g., wavelength and lifetime) may require increasedeffort and complexity of the fluorescent marker and/or integrateddevice. The inventors have recognized, however, that certain types ofinformation may be obtained without increasing the complexity of theintegrated device, as described herein. For example, pulse durationinformation, interpulse duration information, and pulse intensityinformation may be obtained using an existing device configured forobtaining wavelength information and/or lifetime information withoutminimal to no changes to the device required. Thus, the inventors haverecognized that increasing the dimensionality of a discriminationtechnique by using pulse intensity information, pulse durationinformation, and/or interpulse duration information to identify a samplemay improve identification accuracy without significantly increasing thecomplexity of the integrated device.

As described herein, the characteristics described above, e.g.,wavelength information, intensity information, lifetime information,pulse duration information, and interpulse duration information, may beobtained based charge carriers stored in at least one charge storageregion. A component, which, in some embodiments, is part of theintegrated device, is configured for obtaining the information regardingone or more of the characteristics described herein. For example, thecomponent may be a hardware module (e.g. one or more controllers, one ormore processors, circuitry implemented via one or more FieldProgrammable Gate Arrays (FPGAs), an application-specific integratedcircuit (ASICs)) and/or any other suitable component configured toperform the functions of the component described herein.

In some embodiments, the integrated device is configured having onecomponent capable of obtaining wavelength information, intensityinformation, lifetime information, pulse duration information, andinterpulse duration information. In some embodiments, the integrateddevice may be configured having multiple components capable of obtainingwavelength information, intensity information, lifetime information,pulse duration information, and interpulse duration information. Forexample, in some embodiments, each type of information (e.g., wavelengthinformation, lifetime information, etc.) may be obtained by a differentcomponent. In some embodiments, a first component may be configured toobtain some but not all types of information, and one or more othercomponents may be configured to obtain the other types of informationnot obtained by the first component. For example, interpulse durationinformation and pulse duration information may be obtained by a firstcomponent, while wavelength information, luminescence lifetimeinformation, and intensity information may be obtained by one or moreother components. In some embodiments, interpulse duration information,pulse duration information, and luminescence lifetime information may beobtained by a first component, and wavelength information andluminescence lifetime information may be obtained by one or more othercomponents. In some embodiments, multiple components may be configuredto obtain a single type of information (e.g., wavelength information).

The inventors have appreciated that the multi-dimensionaldiscriminations techniques can be implemented in variety ofapplications, two non-limiting examples of which include DNA and/or RNAsequencing applications and/or protein sequencing applications, each ofwhich is described further herein.

a. DNA and/or RNA Sequencing Applications

The inventors have recognized that, in some embodiments, the techniquesdescribed herein for multi-dimensional discrimination of a sample may beused in DNA and/or RNA sequencing applications, as one non-limitingexample. For example, an analytic system described herein may include anintegrated device and an instrument configured to interface with theintegrated device. The integrated device may include an array of pixels,where a pixel includes a reaction chamber and at least onephotodetector. A surface of the integrated device may have a pluralityof reaction chambers, where a reaction chamber is configured to receivea sample from a suspension placed on the surface of the integrateddevice. A suspension may contain multiple samples of a same type, and insome embodiments, different types of samples. In this regard, the phrase“sample of interest” as used herein can refer to a plurality of samplesof a same type that are dispersed in a suspension, for example.Similarly, the phrase “molecule of interest” as used herein can refer toa plurality of molecules of a same type that are dispersed in asuspension. The plurality of reaction chambers may have a suitable sizeand shape such that at least a portion of the reaction chambers receiveone sample from a suspension. In some embodiments, the number of sampleswithin a reaction chamber may be distributed among the reaction chamberssuch that some reaction chambers contain one sample with others containzero, two or more samples.

In some embodiments, a suspension may contain multiple single-strandedDNA templates, and individual reaction chambers on a surface of anintegrated device may be sized and shaped to receive a sequencingtemplate. Sequencing templates may be distributed among the reactionchambers of the integrated device such that at least a portion of thereaction chambers of the integrated device contain a sequencingtemplate. The suspension may also contain labeled nucleotides which thenenter in the reaction chamber and may allow for identification of anucleotide as it is incorporated into a strand of DNA complementary tothe single-stranded DNA template in the reaction chamber. In someembodiments, the suspension may contain sequencing templates and labelednucleotides may be subsequently introduced to a reaction chamber asnucleotides are incorporated into a complementary strand within thereaction chamber. In this manner, timing of incorporation of nucleotidesmay be controlled by when labeled nucleotides are introduced to thereaction chambers of an integrated device.

Excitation light is provided from an excitation source located separatefrom the pixel array of the integrated device. The excitation light isdirected at least in part by elements of the integrated device towardsone or more pixels to illuminate an illumination region within thereaction chamber. A marker may then emit emission light when locatedwithin the illumination region and in response to being illuminated byexcitation light. In some embodiments, one or more excitation sourcesare part of the instrument of the system where components of theinstrument and the integrated device are configured to direct theexcitation light towards one or more pixels.

Emission light emitted from a reaction chamber (e.g., by a fluorescentlabel) may then be detected by one or more photodetectors within a pixelof the integrated device. Characteristics of the detected emission lightmay provide an indication for identifying the marker associated with theemission light. Such characteristics may include any suitable type ofcharacteristic, including an arrival time of photons detected by aphotodetector, an amount of photons accumulated over time by aphotodetector, and/or a distribution of photons across two or morephotodetectors. In some embodiments, a photodetector may have aconfiguration that allows for the detection of one or morecharacteristics associated with emission light, such as timingcharacteristics (e.g., fluorescence lifetime), wavelength, pulseduration, interpulse duration, and/or intensity. As one example, thephotodetector may detect a distribution of photon arrival times after apulse of excitation light propagates through the integrated device, andthe distribution of arrival times may provide an indication of a timingcharacteristic of the emission light (e.g., a proxy for fluorescencelifetime, pulse duration, and/or interpulse duration). In someembodiments, the one or more photodetectors provide an indication of theprobability of emission light emitted by the marker (e.g., fluorescenceintensity). In some embodiments, a plurality of photodetectors may besized and arranged to capture a spatial distribution of the emissionlight (e.g., wavelength). Output signals from the one or morephotodetectors may then be used to distinguish a marker from among aplurality of markers, where the plurality of markers may be used toidentify a sample or its structure. In some embodiments, a sample may beexcited by multiple excitation energies, and emission light and/ortiming characteristics of the emission light from the reaction chamberin response to the multiple excitation energies may distinguish a markerfrom a plurality of markers.

In some embodiments, a system and integrated device similar to thetechnology previously described herein may be implemented to facilitateDNA and/or RNA sequencing applications. For example, a schematicoverview of the system 5-100 is illustrated in FIG. 5-1A. The systemcomprises both an integrated device 5-102 that interfaces with aninstrument 5-104. In some embodiments, instrument 5-104 may include oneor more excitation sources 5-106 integrated as part of instrument 5-104.In some embodiments, an excitation source may be external to bothinstrument 5-104 and integrated device 5-102, and instrument 5-104 maybe configured to receive excitation light from the excitation source anddirect excitation light to the integrated device. The integrated devicemay interface with the instrument using any suitable socket forreceiving the integrated device and holding it in precise opticalalignment with the excitation source. The excitation source 5-106 may beconfigured to provide excitation light to the integrated device 5-102.As illustrated schematically in FIG. 5-1A, the integrated device 5-102has a plurality of pixels 5-112, where at least a portion of pixels mayperform independent analysis of a sample of interest. Such pixels 5-112may be referred to as “passive source pixels” since a pixel receivesexcitation light from a source 5-106 separate from the pixel, whereexcitation light from the source excites some or all of the pixels5-112. Excitation source 5-106 may be any suitable light source.Examples of suitable excitation sources are described in U.S. patentapplication Ser. No. 14/821,688, filed Aug. 7, 2015, titled “INTEGRATEDDEVICE FOR PROBING, DETECTING AND ANALYZING MOLECULES,” under AttorneyDocket No/R0708.70004US02 which is hereby incorporated by reference inits entirety. In some embodiments, excitation source 5-106 includesmultiple excitation sources that are combined to deliver excitationlight to integrated device 5-102. The multiple excitation sources may beconfigured to produce multiple excitation energies or wavelengths.

A pixel 5-112 has a reaction chamber 5-108 configured to receive asingle sample of interest and a photodetector 5-110 for detectingemission light emitted from the reaction chamber in response toilluminating the sample and at least a portion of the reaction chamber5-108 with excitation light provided by the excitation source 5-106. Insome embodiments, reaction chamber 5-108 may retain the sample inproximity to a surface of integrated device 5-102, which may easedelivery of excitation light to the sample and detection of emissionlight from the sample or a reaction component (e.g., a labelednucleotide).

Optical elements for coupling excitation light from excitation lightsource 5-106 to integrated device 5-102 and guiding excitation light tothe reaction chamber 5-108 are located both on integrated device 5-102and the instrument 5-104. Source-to-chamber optical elements maycomprise one or more grating couplers located on integrated device5-102to couple excitation light to the integrated device and waveguides todeliver excitation light from instrument 5-104 to reaction chambers inpixels 5-112. One or more optical splitter elements may be positionedbetween a grating coupler and the waveguides. The optical splitter maycouple excitation light from the grating coupler and deliver excitationlight to at least one of the waveguides. In some embodiments, theoptical splitter may have a configuration that allows for delivery ofexcitation light to be substantially uniform across all the waveguidessuch that each of the waveguides receives a substantially similar amountof excitation light. Such embodiments may improve performance of theintegrated device by improving the uniformity of excitation lightreceived by reaction chambers of the integrated device.

Reaction chamber 5-108, a portion of the excitation source-to-chamberoptics, and the reaction chamber-to-photodetector optics are located onintegrated device 5-102. Excitation source 5-106 and a portion of thesource-to-chamber components are located in instrument 5-104. In someembodiments, a single component may play a role in both couplingexcitation light to reaction chamber 5-108 and delivering emission lightfrom reaction chamber to photodetector 5-110. Examples of suitablecomponents, for coupling excitation light to a reaction chamber and/ordirecting emission light to a photodetector, to include in an integrateddevice are described in U.S. patent application Ser. No. 14/821,688,filed Aug. 7, 2015, titled “INTEGRATED DEVICE FOR PROBING, DETECTING ANDANALYZING MOLECULES,” under Attorney Docket Ser. No. 14/821,688 and U.S.patent application Ser. No. 14/543,865, filed Nov. 17, 2014, titled“INTEGRATED DEVICE WITH EXTERNAL LIGHT SOURCE FOR PROBING, DETECTING,AND ANALYZING MOLECULES,” under Attorney Docket No. R0708.70005US00 bothof which are hereby incorporated by reference in their entirety.

Pixel 5-112 is associated with its own individual reaction chamber 5-108and at least one photodetector 5-110. The plurality of pixels ofintegrated device 5-102 may be arranged to have any suitable shape,size, and/or dimensions. Integrated device 5-102 may have any suitablenumber of pixels. The number of pixels in integrated device 2-102 may bein the range of approximately 10,000 pixels to 1,000,000 pixels or anyvalue or range of values within that range. In some embodiments, thepixels may be arranged in an array of 512 pixels by 512 pixels.Integrated device 5-102 may interface with instrument 5-104 in anysuitable manner. In some embodiments, instrument 5-104 may have aninterface that detachably couples to integrated device 5-102 such that auser may attach integrated device 5-102 to instrument 5-104 for use ofintegrated device 5-102 to analyze at least one sample of interest in asuspension and remove integrated device 5-102 from instrument 5-104 toallow for another integrated device to be attached. The interface ofinstrument 5-104 may position integrated device 5-102 to couple withcircuitry of instrument 5-104 to allow for readout signals from one ormore photodetectors to be transmitted to instrument 5-104. Integrateddevice 5-102 and instrument 5-104 may include multi-channel, high-speedcommunication links for handling data associated with large pixel arrays(e.g., more than 10,000 pixels).

A cross-sectional schematic of integrated device 5-102 illustrating arow of pixels is shown in FIG. 5-1B. Integrated device 5-102 may includecoupling region 5-201, routing region 5-202, and pixel region 5-203.Pixel region 5-203 may include a plurality of pixels 5-112 havingreaction chambers 5-108 positioned on a surface at a location separatefrom coupling region 5-201, which is where excitation light (shown asthe dashed arrow) couples to integrated device 5-102. Reaction chambers5-108 may be formed through metal layer(s) 5-116. One pixel 5-112,illustrated by the dotted rectangle, is a region of integrated device5-102 that includes a reaction chamber 5-108 and a photodetection regionhaving one or more photodetectors 5-110.

FIG. 5-1B illustrates the path of excitation (shown in dashed lines) bycoupling a beam of excitation light to coupling region 5-201 and toreaction chambers 5-108. The row of reaction chambers 5-108 shown inFIG. 5-1B may be positioned to optically couple with waveguide 5-220.Excitation light may illuminate a sample located within a reactionchamber. The sample or a reaction component (e.g., fluorescent label)may reach an excited state in response to being illuminated by theexcitation light. When in an excited state, the sample or reactioncomponent may emit emission light, which may be detected by one or morephotodetectors associated with the reaction chamber. FIG. 5-1Bschematically illustrates the path of emission light (shown as the solidline) from a reaction chamber 5-108 to photodetector(s) 5-110 of pixel5-112. The photodetector(s) 5-110 of pixel 5-112 may be configured andpositioned to detect emission light from reaction chamber 5-108.Examples of suitable photodetectors are described in U.S. patentapplication Ser. No. 14/821,656, filed Aug. 7, 2015, titled “INTEGRATEDDEVICE FOR TEMPORAL BINNING OF RECEIVED PHOTONS,” under Attorney DocketNo. R0708.70002US02 which is hereby incorporated by reference in itsentirety. For an individual pixel 5-112, a reaction chamber 5-108 andits respective photodetector(s) 5-110 may be aligned along a common axis(along the y-direction shown in FIG. 5-1B). In this manner, thephotodetector(s) may overlap with the reaction chamber within a pixel5-112.

The directionality of the emission light from a reaction chamber 5-108may depend on the positioning of the sample in the reaction chamber5-108 relative to metal layer(s) because metal layer(s) 5-116 may act toreflect emission light. In this manner, a distance between metallayer(s) 5-116 and a fluorescent marker positioned in a reaction chamber5-108 may impact the efficiency of photodetector(s) 5-110, that are inthe same pixel as the reaction chamber, to detect the light emitted bythe fluorescent marker. The distance between metal layer(s) 5-116 andthe bottom surface of a reaction chamber 5-106, which is proximate towhere a sample may be positioned during operation, may be in the rangeof 100 nm to 500 nm, or any value or range of values in that range. Insome embodiments the distance between metal layer(s) and the bottomsurface of a reaction chamber 5-108 is approximately 300 nm.

The distance between the sample and the photodetector(s) may also impactefficiency in detecting emission light. By decreasing the distance lighthas to travel between the sample and the photodetector(s), detectionefficiency of emission light may be improved. In addition, smallerdistances between the sample and the photodetector(s) may allow forpixels that occupy a smaller area footprint of the integrated device,which can allow for a higher number of pixels to be included in theintegrated device. The distance between the bottom surface of a reactionchamber 5-108 and photodetector(s) may be in the range of 1 μm to 15 μm,or any value or range of values in that range.

Photonic structure(s) 5-230 may be positioned between reaction chambers5-108 and photodetectors 5-110 and configured to reduce or preventexcitation light from reaching photodetectors 5-110, which may otherwisecontribute to signal noise in detecting emission light. As shown in FIG.5-1B, the one or more photonic structures 5-230 may be positionedbetween waveguide 5-220 and photodetectors 5-110. Photonic structure(s)5-230 may include one or more optical rejection photonic structuresincluding a spectral filter, a polarization filter, and a spatialfilter. Photonic structure(s) 5-230 may be positioned to align withindividual reaction chambers 5-108 and their respective photodetector(s)5-110 along a common axis. Metal layers 5-240, which may act as acircuitry for integrated device 5-102, may also act as a spatial filter,in accordance with some embodiments. In such embodiments, one or moremetal layers 5-240 may be positioned to block some or all excitationlight from reaching photodetector(s) 5-110.

Coupling region 5-201 may include one or more optical componentsconfigured to couple excitation light from an external excitationsource. Coupling region 5-201 may include grating coupler 5-216positioned to receive some or all of a beam of excitation light.Examples of suitable grating couplers are described in U.S. patentapplication Ser. No. 15/844,403, filed Dec. 15, 2017, titled “OPTICALCOUPLER AND WAVEGUIDE SYSTEM,” under Attorney Docket No. R0708.70021US01which is hereby incorporated by reference in its entirety. Gratingcoupler 5-216 may couple excitation light to waveguide 5-220, which maybe configured to propagate excitation light to the proximity of one ormore reaction chambers 5-108. Alternatively, coupling region 5-201 maycomprise other well-known structures for coupling light into awaveguide.

Components located off of the integrated device may be used to positionand align the excitation source 5-106 to the integrated device. Suchcomponents may include optical components including lenses, mirrors,prisms, windows, apertures, attenuators, and/or optical fibers.Additional mechanical components may be included in the instrument toallow for control of one or more alignment components. Such mechanicalcomponents may include actuators, stepper motors, and/or knobs. Examplesof suitable excitation sources and alignment mechanisms are described inU.S. patent application Ser. No. 15/161,088, filed May 20, 2016, titled“PULSED LASER AND SYSTEM,” under Attorney Docket No. R0708.70010US02which is hereby incorporated by reference in its entirety. Anotherexample of a beam-steering module is described in U.S. patentapplication Ser. No. 15/842,720, filed Dec. 14, 2017, titled “COMPACTBEAM SHAPING AND STEERING ASSEMBLY,” under Attorney Docket No.R0708.70024US01 which is hereby incorporated herein by reference.

A sample to be analyzed may be introduced into reaction chamber 5-108 ofpixel 5-112. The sample may be a biological sample or any other suitablesample, such as a chemical sample. In some cases, the suspension mayinclude multiple molecules of interest and the reaction chamber may beconfigured to isolate a single molecule. In some instances, thedimensions of the reaction chamber may act to confine a single moleculewithin the reaction chamber, allowing measurements to be performed onthe single molecule. Excitation light may be delivered into the reactionchamber 5-108, so as to excite the sample or at least one fluorescentmarker attached to the sample or otherwise associated with the samplewhile it is within an illumination area within the reaction chamber5-108.

In operation, parallel analyses of samples within the reaction chambersare carried out by exciting some or all of the samples within thereaction chambers using excitation light and detecting signals with thephotodetectors that are representative of emission light from thereaction chambers. Emission light from a sample or reaction component(e.g., fluorescent label) may be detected by a correspondingphotodetector and converted to at least one electrical signal. Theelectrical signals may be transmitted along conducting lines (e.g.,metal layers 5-240) in the circuitry of the integrated device, which maybe connected to an instrument interfaced with the integrated device. Theelectrical signals may be subsequently processed and/or analyzed.Processing or analyzing of electrical signals may occur on a suitablecomputing device either located on or off the instrument.

Instrument 5-104 may include a user interface for controlling operationof instrument 5-104 and/or integrated device 5-102. The user interfacemay be configured to allow a user to input information into theinstrument, such as commands and/or settings used to control thefunctioning of the instrument. In some embodiments, the user interfacemay include buttons, switches, dials, and a microphone for voicecommands. The user interface may allow a user to receive feedback on theperformance of the instrument and/or integrated device, such as properalignment and/or information obtained by readout signals from thephotodetectors on the integrated device. In some embodiments, the userinterface may provide feedback using a speaker to provide audiblefeedback. In some embodiments, the user interface may include indicatorlights and/or a display screen for providing visual feedback to a user.

In some embodiments, instrument 5-104 may include a computer interfaceconfigured to connect with a computing device. Computer interface may bea USB interface, a FireWire interface, or any other suitable computerinterface. Computing device may be any general purpose computer, such asa laptop or desktop computer. In some embodiments, computing device maybe a server (e.g., cloud-based server) accessible over a wirelessnetwork via a suitable computer interface. The computer interface mayfacilitate communication of information between instrument 5-104 and thecomputing device. Input information for controlling and/or configuringthe instrument 5-104 may be provided to the computing device andtransmitted to instrument 5-104 via the computer interface. Outputinformation generated by instrument 5-104 may be received by thecomputing device via the computer interface. Output information mayinclude feedback about performance of instrument 5-104, performance ofintegrated device 5-112, and/or data generated from the readout signalsof photodetector 5-110.

In some embodiments, instrument 5-104 may include a processing deviceconfigured to analyze data received from one or more photodetectors ofintegrated device 5-102 and/or transmit control signals to excitationsource(s) 2-106. In some embodiments, the processing device may comprisea general purpose processor, a specially-adapted processor (e.g., acentral processing unit (CPU) such as one or more microprocessor ormicrocontroller cores, a field-programmable gate array (FPGA), anapplication-specific integrated circuit (ASIC), a custom integratedcircuit, a digital signal processor (DSP), or a combination thereof.) Insome embodiments, the processing of data from one or more photodetectorsmay be performed by both a processing device of instrument 5-104 and anexternal computing device. In other embodiments, an external computingdevice may be omitted and processing of data from one or morephotodetectors may be performed solely by a processing device ofintegrated device 5-102.

Referring to FIG. 5-1C, a portable, advanced analytic instrument 5-100can comprise one or more pulsed optical sources 5-106 mounted as areplaceable module within, or otherwise coupled to, the instrument5-100. The portable analytic instrument 5-100 can include an opticalcoupling system 5-115 and an analytic system 5-160. The optical couplingsystem 5-115 can include some combination of optical components (whichmay include, for example, none, one from among, or more than onecomponent from among the following components: lens, mirror, opticalfilter, attenuator, beam-steering component, beam shaping component) andbe configured to operate on and/or couple output optical pulses 5-122from the pulsed optical source 5-106 to the analytic system 5-160. Theanalytic system 5-160 can include a plurality of components that arearranged to direct the optical pulses to at least one reaction chamberfor sample analysis, receive one or more optical signals (e.g.,fluorescence, backscattered radiation) from the at least one reactionchamber, and produce one or more electrical signals representative ofthe received optical signals. In some embodiments, the analytic system5-160 can include one or more photodetectors and may also includesignal-processing electronics (e.g., one or more microcontrollers, oneor more field-programmable gate arrays, one or more microprocessors, oneor more digital signal processors, logic gates, etc.) configured toprocess the electrical signals from the photodetectors. The analyticsystem 5-160 can also include data transmission hardware configured totransmit and receive data to and from external devices (e.g., one ormore external devices on a network to which the instrument 5-100 canconnect via one or more data communications links). In some embodiments,the analytic system 5-160 can be configured to receive abio-optoelectronic chip 5-140, which holds one or more samples to beanalyzed.

FIG. 5-1D depicts a further detailed example of a portable analyticalinstrument 5-100 that includes a compact pulsed optical source 5-108. Inthis example, the pulsed optical source 5-108 comprises a compact,passively mode-locked laser module 5-113. A passively mode-locked lasercan produce optical pulses autonomously, without the application of anexternal pulsed signal. In some implementations, the module can bemounted to an instrument chassis or frame 5-103, and may be locatedinside an outer casing of the instrument. According to some embodiments,a pulsed optical source 5-106 can include additional components that canbe used to operate the optical source and operate on an output beam fromthe optical source 5-106. A mode-locked laser 5-113 may comprise anelement (e.g., saturable absorber, acousto-optic modulator, Kerr lens)in a laser cavity, or coupled to the laser cavity, that induces phaselocking of the laser's longitudinal frequency modes. The laser cavitycan be defined in part by cavity end minors 5-111, 5-119. Such lockingof the frequency modes results in pulsed operation of the laser (e.g.,an intracavity pulse 5-120 bounces back-and-forth between the cavity endminors) and produces a stream of output optical pulses 5-122 from oneend mirror 5-111 which is partially transmitting.

In some cases, the analytic instrument 5-100 is configured to receive aremovable, packaged, bio-optoelectronic or optoelectronic chip 5-140(also referred to as a “disposable chip”). The disposable chip caninclude a bio-optoelectronic chip, for example, that comprises aplurality of reaction chambers, integrated optical components arrangedto deliver optical excitation energy to the reaction chambers, andintegrated photodetectors arranged to detect fluorescent emission fromthe reaction chambers. In some implementations, the chip 5-140 can bedisposable after a single use, whereas in other implementations the chip5-140 can be reused two or more times. When the chip 5-140 is receivedby the instrument 5-100, it can be in electrical and opticalcommunication with the pulsed optical source 5-106 and with apparatus inthe analytic system 5-160. Electrical communication may be made throughelectrical contacts on the chip package, for example.

In some embodiments and referring to FIG. 5-1D, the disposable chip5-140 can be mounted (e.g., via a socket connection) on an electroniccircuit board 5-130, such as a printed circuit board (PCB) that caninclude additional instrument electronics. For example, the PCB 5-130can include circuitry configured to provide electrical power, one ormore clock signals, and control signals to the optoelectronic chip5-140, and signal-processing circuitry arranged to receive signalsrepresentative of fluorescent emission detected from the reactionchambers. Data returned from the optoelectronic chip can be processed inpart or entirely by electronics on the instrument 5-100, although datamay be transmitted via a network connection to one or more remote dataprocessors, in some implementations. The PCB 5-130 can also includecircuitry configured to receive feedback signals from the chip relatingto optical coupling and power levels of the optical pulses 5-122 coupledinto waveguides of the optoelectronic chip 5-140. The feedback signalscan be provided to one or both of the pulsed optical source 5-106 andoptical system 5-115 to control one or more parameters of the outputbeam of optical pulses 5-122. In some cases, the PCB 5-130 can provideor route power to the pulsed optical source 5-106 for operating theoptical source and related circuitry in the optical source 5-106.

According to some embodiments, the pulsed optical source 5-106 comprisesa compact mode-locked laser module 5-113. The mode-locked laser cancomprise a gain medium 5-105 (which can be solid-state material in someembodiments), an output coupler 5-111, and a laser-cavity end minor5-119. The mode-locked laser's optical cavity can be bound by the outputcoupler 5-111 and end minor 5-119. An optical axis 5-125 of the lasercavity can have one or more folds (turns) to increase the length of thelaser cavity and provide a desired pulse repetition rate. The pulserepetition rate is determined by the length of the laser cavity (e.g.,the time for an optical pulse to make a round-trip within the lasercavity).

In some embodiments, there can be additional optical elements (not shownin FIG. 5-1D) in the laser cavity for beam shaping, wavelengthselection, and/or pulse forming. In some cases, the end mirror 5-119comprises a saturable-absorber mirror (SAM) that induces passive modelocking of longitudinal cavity modes and results in pulsed operation ofthe mode-locked laser. The mode-locked laser module 5-113 can furtherinclude a pump source (e.g., a laser diode, not shown in FIG. 5-1D) forexciting the gain medium 5-105. Further details of a mode-locked lasermodule 5-113 can be found in U.S. patent application Ser. No.15/844,469, titled “Compact Mode-Locked Laser Module,” filed Dec. 15,2017, each application of which is incorporated herein by reference.

When the laser 5-113 is mode locked, an intracavity pulse 5-120 cancirculate between the end minor 5-119 and the output coupler 5-111, anda portion of the intracavity pulse can be transmitted through the outputcoupler 5-111 as an output pulse 5-122. Accordingly, a train of outputpulses 5-122, as depicted in the graph of FIG. 5-2 , can be detected atthe output coupler as the intracavity pulse 5-120 bounces back-and-forthbetween the output coupler 5-111 and end minor 5-119 in the lasercavity.

FIG. 5-2 depicts temporal intensity profiles of the output pulses 5-122,though the illustration is not to scale. In some embodiments, the peakintensity values of the emitted pulses may be approximately equal, andthe profiles may have a Gaussian temporal profile, though other profilessuch as a sech2 profile may be possible. In some cases, the pulses maynot have symmetric temporal profiles and may have other temporal shapes.The duration of each pulse may be characterized by afull-width-half-maximum (FWHM) value, as indicated in FIG. 5-2 .According to some embodiments of a mode-locked laser, ultrashort opticalpulses can have FWHM values less than 100 picoseconds (ps). In somecases, the FWHM values can be between approximately 5 ps andapproximately 30 ps.

The output pulses 5-122 can be separated by regular intervals T. Forexample, T can be determined by a round-trip travel time between theoutput coupler 5-111 and cavity end minor 5-119. According to someembodiments, the pulse-separation interval T can be between about 1 nsand about 30 ns. In some cases, the pulse-separation interval T can bebetween about ns and about 20 ns, corresponding to a laser-cavity length(an approximate length of the optical axis 5-125 within the lasercavity) between about 0.7 meter and about 3 meters. In embodiments, thepulse-separation interval corresponds to a round trip travel time in thelaser cavity, so that a cavity length of 3 meters (round-trip distanceof 6 meters) provides a pulse-separation interval T of approximately 20ns.

According to some embodiments, a desired pulse-separation interval T andlaser-cavity length can be determined by a combination of the number ofreaction chambers on the chip 5-140, fluorescent emissioncharacteristics, and the speed of data-handling circuitry for readingdata from the optoelectronic chip 5-140. In embodiments, differentfluorophores can be distinguished by their different fluorescent decayrates or characteristic lifetimes. Accordingly, there needs to be asufficient pulse-separation interval T to collect adequate statisticsfor the selected fluorophores to distinguish between their differentdecay rates. Additionally, if the pulse-separation interval T is tooshort, the data handling circuitry cannot keep up with the large amountof data being collected by the large number of reaction chambers.Pulse-separation interval T between about 5 ns and about 20 ns issuitable for fluorophores that have decay rates up to about 2 ns and forhandling data from between about 60,000 and 10,000,000 reactionchambers.

According to some implementations, a beam-steering module 5-150 canreceive output pulses from the pulsed optical source 5-106 and isconfigured to adjust at least the position and incident angles of theoptical pulses onto an optical coupler (e.g., grating coupler) of theoptoelectronic chip 5-140. In some cases, the output pulses 5-122 fromthe pulsed optical source 5-106 can be operated on by a beam-steeringmodule 5-150 to additionally or alternatively change a beam shape and/orbeam rotation at an optical coupler on the optoelectronic chip 5-140. Insome implementations, the beam-steering module 5-150 can further providefocusing and/or polarization adjustments of the beam of output pulsesonto the optical coupler. One example of a beam-steering module isdescribed in U.S. patent application Ser. No. 15/161,088 titled “PulsedLaser and Bioanalytic System,” filed May 20, 2016, which is incorporatedherein by reference. Another example of a beam-steering module isdescribed in a separate U.S. patent application No. 62/435,679, filedDec. 16, 2016 and titled “Compact Beam Shaping and Steering Assembly,”which is incorporated herein by reference.

Referring to FIG. 5-3 , the output pulses 5-122 from a pulsed opticalsource can be coupled into one or more optical waveguides 5-312 on abio-optoelectronic chip 5-140, for example. In some embodiments, theoptical pulses can be coupled to one or more waveguides via a gratingcoupler 5-310, though coupling to an end of one or more opticalwaveguides on the optoelectronic chip can be used in some embodiments.According to some embodiments, a quad detector 5-320 can be located on asemiconductor substrate 5-305 (e.g., a silicon substrate) for aiding inalignment of the beam of optical pulses 5-122 to a grating coupler5-310. The one or more waveguides 5-312 and reaction chambers orreaction chambers 5-330 can be integrated on the same semiconductorsubstrate with intervening dielectric layers (e.g., silicon dioxidelayers) between the substrate, waveguide, reaction chambers, andphotodetectors 5-322.

Each waveguide 5-312 can include a tapered portion 5-315 below thereaction chambers 5-330 to equalize optical power coupled to thereaction chambers along the waveguide. The reducing taper can force moreoptical energy outside the waveguide's core, increasing coupling to thereaction chambers and compensating for optical losses along thewaveguide, including losses for light coupling into the reactionchambers. A second grating coupler 5-317 can be located at an end ofeach waveguide to direct optical energy to an integrated photodiode5-324. The integrated photodiode can detect an amount of power coupleddown a waveguide and provide a detected signal to feedback circuitrythat controls the beam-steering module 5-150, for example.

The reaction chambers 5-330 or reaction chambers 5-330 can be alignedwith the tapered portion 5-315 of the waveguide and recessed in a tub5-340. There can be photodetectors 5-322 located on the semiconductorsubstrate 5-305 for each reaction chamber 5-330. In some embodiments, asemiconductor absorber (shown in FIG. 5-5 as an optical filter 5-530)may be located between the waveguide and a photodetector 5-322 at eachpixel. A metal coating and/or multilayer coating 5-350 can be formedaround the reaction chambers and above the waveguide to prevent opticalexcitation of fluorophores that are not in the reaction chambers (e.g.,dispersed in a solution above the reaction chambers). The metal coatingand/or multilayer coating 5-350 may be raised beyond edges of the tub5-340 to reduce absorptive losses of the optical energy in the waveguide5-312 at the input and output ends of each waveguide.

There can be a plurality of rows of waveguides, reaction chambers, andtime-binning photodetectors on the optoelectronic chip 5-140. Forexample, there can be 128 rows, each having 512 reaction chambers, for atotal of 65,536 reaction chambers in some implementations. Otherimplementations may include fewer or more reaction chambers, and mayinclude other layout configurations. Optical power from the pulsedoptical source 5-106 can be distributed to the multiple waveguides viaone or more star couplers or multi-mode interference couplers, or by anyother means, located between an optical coupler 5-310 to the chip 5-140and the plurality of waveguides 5-312.

FIG. 5-4 illustrates optical energy coupling from an optical pulse 5-122within a tapered portion of waveguide 5-315 to a reaction chamber 5-330.The drawing has been produced from an electromagnetic field simulationof the optical wave that accounts for waveguide dimensions, reactionchamber dimensions, the different materials' optical properties, and thedistance of the tapered portion of waveguide 5-315 from the reactionchamber 5-330. The waveguide can be formed from silicon nitride in asurrounding medium 5-410 of silicon dioxide, for example. The waveguide,surrounding medium, and reaction chamber can be formed bymicrofabrication processes described in U.S. application Ser. No.14/821,688, filed Aug. 7, 2015, titled “Integrated Device for Probing,Detecting and Analyzing Molecules.” According to some embodiments, anevanescent optical field 5-420 couples optical energy transported by thewaveguide to the reaction chamber 5-330.

A non-limiting example of a biological reaction taking place in areaction chamber 5-330 is depicted in FIG. 5-5 . The example depictssequential incorporation of nucleotides or nucleotide analogs into agrowing strand that is complementary to a target nucleic acid. Thesequential incorporation can take place in a reaction chamber 5-330, andcan be detected by an advanced analytic instrument to sequence DNA. Thereaction chamber can have a depth between about 150 nm and about 250 nmand a diameter between about 80 nm and about 160 nm. A metallizationlayer 5-540 (e.g., a metallization for an electrical referencepotential) can be patterned above a photodetector 5-322 to provide anaperture or iris that blocks stray light from adjacent reaction chambersand other unwanted light sources. According to some embodiments,polymerase 5-520 can be located within the reaction chamber 5-330 (e.g.,attached to a base of the chamber). The polymerase can take up a targetnucleic acid 5-510 (e.g., a portion of nucleic acid derived from DNA),and sequence a growing strand of complementary nucleic acid to produce agrowing strand of DNA 5-512. Nucleotides or nucleotide analogs labeledwith different fluorophores can be dispersed in a solution above andwithin the reaction chamber.

When a labeled nucleotide or nucleotide analog 5-610 is incorporatedinto a growing strand of complementary nucleic acid, as depicted in FIG.5-6 , one or more attached fluorophores 5-630 can be repeatedly excitedby pulses of optical energy coupled into the reaction chamber 5-330 fromthe waveguide 5-315. In some embodiments, the fluorophore orfluorophores 5-630 can be attached to one or more nucleotides ornucleotide analogs 5-610 with any suitable linker 5-620. Anincorporation event may last for a period of time up to about 100 ms.During this time, pulses of fluorescent emission resulting fromexcitation of the fluorophore(s) by pulses from the mode-locked lasercan be detected with a time-binning photodetector 5-322, for example. Insome embodiments, there can be one or more additional integratedelectronic devices 5-323 at each pixel for signal handling (e.g.,amplification, read-out, routing, signal preprocessing, etc.). Accordingto some embodiments, each pixel can include at least one optical filter5-530 (e.g., a semiconductor absorber) that passes fluorescent emissionand reduces transmission of radiation from the excitation pulse. Someimplementations may not use the optical filter 5-530. By attachingfluorophores with different emission characteristics (e.g., fluorescentdecay rates, intensity, fluorescent wavelength, pulse duration,interpulse duration) to the different nucleotides (A,C,G,T), detectingand distinguishing the different emission characteristics while thestrand of DNA 5-512 incorporates a nucleic acid and enablesdetermination of the genetic sequence of the growing strand of DNA.

Techniques for time binning charge carriers generated by incidentemission light to facilitate obtaining timing information of theemission light (e.g., fluorescent lifetime, pulse duration, interpulseduration) described herein, for example with respect to Sections III.And V may be applied to DNA and/or RNA sequencing applications. Forexample, according to some embodiments, an advanced analytic instrument5-100 that is configured to analyze samples based on fluorescentemission characteristics can detect differences in fluorescent lifetimesand/or intensities between different fluorescent molecules, and/ordifferences between lifetimes and/or intensities of the same fluorescentmolecules in different environments. By way of explanation, FIG. 5-7plots two different fluorescent emission probability curves (A and B),which can be representative of fluorescent emission from two differentfluorescent molecules, for example. With reference to curve A (dashedline), after being excited by a short or ultrashort optical pulse, aprobability pA(t) of a fluorescent emission from a first molecule maydecay with time, as depicted. In some cases, the decrease in theprobability of a photon being emitted over time can be represented by anexponential decay function p_A (t)=P_Ao e{circumflex over ( )}(−t/τ_1),where PAo is an initial emission probability and τ1 is a temporalparameter associated with the first fluorescent molecule thatcharacterizes the emission decay probability. τ1 may be referred to asthe “fluorescence lifetime,” “emission lifetime,” or “lifetime” of thefirst fluorescent molecule. In some cases, the value of τ1 can bealtered by a local environment of the fluorescent molecule. Otherfluorescent molecules can have different emission characteristics thanthat shown in curve A. For example, another fluorescent molecule canhave a decay profile that differs from a single exponential decay, andits lifetime can be characterized by a half-life value or some othermetric.

A second fluorescent molecule may have a decay profile pB(t) that isexponential, but has a measurably different lifetime τ2, as depicted forcurve B in FIG. 5-7 . In the example shown, the lifetime for the secondfluorescent molecule of curve B is shorter than the lifetime for curveA, and the probability of emission pB(t) is higher sooner afterexcitation of the second molecule than for curve A. Differentfluorescent molecules can have lifetimes or half-life values rangingfrom about 0.1 ns to about 20 ns, in some embodiments.

Differences in fluorescent emission lifetimes can be used to discernbetween the presence or absence of different fluorescent moleculesand/or to discern between different environments or conditions to whicha fluorescent molecule is subjected. In some cases, discerningfluorescent molecules based on lifetime (rather than emissionwavelength, for example) can simplify aspects of an analyticalinstrument 5-100. As an example, wavelength-discriminating optics (suchas wavelength filters, dedicated detectors for each wavelength,dedicated pulsed optical sources at different wavelengths, and/ordiffractive optics) can be reduced in number or eliminated whendiscerning fluorescent molecules based on lifetime. In some cases, asingle pulsed optical source operating at a single characteristicwavelength can be used to excite different fluorescent molecules thatemit within a same wavelength region of the optical spectrum but havemeasurably different lifetimes. An analytic system that uses a singlepulsed optical source, rather than multiple sources operating atdifferent wavelengths, to excite and discern different fluorescentmolecules emitting in a same wavelength region can be less complex tooperate and maintain, more compact, and can be manufactured at lowercost.

Although analytic systems based on fluorescent lifetime analysis canhave certain benefits, the amount of information obtained by an analyticsystem and/or detection accuracy can be increased by allowing foradditional detection techniques. For example, some analytic systems5-160 can additionally be configured to discern one or more propertiesof a sample based on fluorescent wavelength, pulse duration/width,interpulse duration, and/or fluorescent intensity as described herein.

Referring again to FIG. 5-7 , according to some embodiments, differentfluorescent lifetimes can be distinguished with a photodetector that isconfigured to time-bin fluorescent emission events following excitationof a fluorescent molecule. The time binning can occur during a singlecharge-accumulation cycle for the photodetector. A charge-accumulationcycle is an interval between read-out events during whichphoto-generated carriers are accumulated in bins of the time-binningphotodetector. The concept of determining fluorescent lifetime bytime-binning of emission events is introduced graphically in FIG. 5-8 .At time t_(e) just prior to t₁, a fluorescent molecule or ensemble offluorescent molecules of a same type (e.g., the type corresponding tocurve B of FIG. 5-7 ) is (are) excited by a short or ultrashort opticalpulse. For a large ensemble of molecules, the intensity of emission canhave a time profile similar to curve B, as depicted in FIG. 5-8 .

For a single molecule or a small number of molecules, however, theemission of fluorescent photons occurs according to the statistics ofcurve B in FIG. 5-7 , for this example. A time-binning photodetector5-322 can accumulate carriers generated from emission events intodiscrete time bins. Three bins are indicated in FIG. 5-8 , though fewerbins or more bins may be used in embodiments. The bins are temporallyresolved with respect to the excitation time t_(e) of the fluorescentmolecule(s). For example, a first bin can accumulate carriers producedduring an interval between times t₁ and t₂, occurring after theexcitation event at time t_(e). A second bin can accumulate carriersproduced during an interval between times t₂ and t₃, and a third bin canaccumulate carriers produced during an interval between times t₃ and t₄.When a large number of emission events are summed, carriers accumulatedin the time bins can approximate the decaying intensity curve shown inFIG. 5-8 , and the binned signals can be used to distinguish betweendifferent fluorescent molecules or different environments in which afluorescent molecule is located.

Examples of a time-binning photodetector 5-322 are described in U.S.patent application Ser. No. 14/821,656, filed Aug. 7, 2015, titled“Integrated Device for Temporal Binning of Received Photons” and in U.S.patent application Ser. No. 15/852,571, filed Dec. 22, 2017, titled“Integrated Photodetector with Direct Binning Pixel,” which are bothincorporated herein by reference in their entirety. For explanationpurposes, a non-limiting embodiment of a time-binning photodetector isdepicted in FIG. 5-9 . A single time-binning photodetector 5-322 cancomprise a photon-absorption/carrier-generation region 5-902, acarrier-discharge channel 5-906, and a plurality of carrier-storageregions 5-908 a, 5-908 b all formed on a semiconductor substrate.Carrier-transport channels 5-907 can connect between thephoton-absorption/carrier-generation region 5-902 and carrier-storageregions 5-908 a, 5-908 b. In the illustrated example, twocarrier-storage regions are shown, but there may be more or fewer. Therecan be a read-out channel 5-910 connected to the carrier-storageregions. The photon-absorption/carrier-generation region 5-902,carrier-discharge channel 5-906, carrier-storage regions 5-908 a, 5-908b, and read-out channel 5-910 can be formed by doping the semiconductorlocally and/or forming adjacent insulating regions to providephotodetection capability, confinement, and transport of carriers. Atime-binning photodetector 5-322 can also include a plurality ofelectrodes 5-920, 5-921, 5-922, 5-923, 5-924 formed on the substratethat are configured to generate electric fields in the device fortransporting carriers through the device.

In operation, a portion of an excitation pulse 5-122 from a pulsedoptical source 5-106 (e.g., a mode-locked laser) is delivered to areaction chamber 5-330 over the time-binning photodetector 5-322.Initially, some excitation radiation photons 5-901 may arrive at thephoton-absorption/carrier-generation region 5-902 and produce carriers(shown as light-shaded circles). There can also be some fluorescentemission photons 5-903 that arrive with the excitation radiation photons5-901 and produce corresponding carriers (shown as dark-shaded circles).Initially, the number of carriers produced by the excitation radiationcan be too large compared to the number of carriers produced by thefluorescent emission. The initial carriers produced during a timeinterval |t_(e)-t₁| can be rejected by gating them into acarrier-discharge channel 5-906 with a first transfer gate 5-920, forexample.

At a later times mostly fluorescent emission photons 5-903 arrive at thephoton-absorption/carrier-generation region 5-902 and produce carriers(indicated a dark-shaded circles) that provide useful and detectablesignal that is representative of fluorescent emission from the reactionchamber 5-330. According to some detection methods, a second electrode5-921 and third electrode 5-923 can be gated at a later time to directcarriers produced at a later time (e.g., during a second time interval|t₁-t₂|) to a first carrier-storage region 5-908 a. Subsequently, afourth electrode 5-922 and fifth electrode 5-924 can be gated at a latertime (e.g., during a third time interval |t₂-t₃|) to direct carriers toa second carrier-storage region 5-908 b. Charge accumulation cancontinue in this manner after excitation pulses for a large number ofexcitation pulses to accumulate an appreciable number of carriers andsignal level in each carrier-storage region 5-908 a, 5-908 b. At a latertime, the signal can be read out from the bins. In some implementations,the time intervals corresponding to each storage region are at thesub-nanosecond time scale, though longer time scales can be used in someembodiments (e.g., in embodiments where fluorophores have longer decaytimes).

The process of generating and time-binning carriers after an excitationevent (e.g., excitation pulse from a pulsed optical source) can occuronce after a single excitation pulse or be repeated multiple times aftermultiple excitation pulses during a single charge-accumulation cycle forthe time-binning photodetector 5-322. After charge accumulation iscomplete, carriers can be read out of the storage regions via theread-out channel 5-910. For example, an appropriate biasing sequence canbe applied to electrodes 5-923, 5-924 and at least to electrode 5-940 toremove carriers from the storage regions 5-908 a, 5-908 b. The chargeaccumulation and read-out processes can occur in a massively paralleloperation on the optoelectronic chip 5-140 resulting in frames of data.

Although the described example in connection with FIG. 5-9 includesmultiple charge storage regions 5-908 a, 5-908 b in some cases a singlecharge storage region may be used instead. For example, only bin1 may bepresent in a time-binning photodetector 5-322. In such a case, a singlestorage regions 5-908 a can be operated in a variable time-gated mannerto look at different time intervals after different excitation events.For example, after pulses in a first series of excitation pulses,electrodes for the storage region 5-908 a can be gated to collectcarriers generated during a first time interval (e.g., during the secondtime interval |t₁-t₂|), and the accumulated signal can be read out aftera first predetermined number of pulses. After pulses in a subsequentseries of excitation pulses at the same reaction chamber, the sameelectrodes for the storage region 5-908 a can be gated to collectcarriers generated during a different interval (e.g., during the thirdtime interval |t₂-t₃|), and the accumulated signal can be read out aftera second predetermined number of pulses. Carriers could be collectedduring later time intervals in a similar manner if needed. In thismanner, signal levels corresponding to fluorescent emission duringdifferent time periods after arrival of an excitation pulse at areaction chamber can be produced using a single carrier-storage region.

Regardless of how charge accumulation is carried out for different timeintervals after excitation, signals that are read out can provide ahistogram of bins that are representative of the fluorescent emissiondecay characteristics, for example. An example process is illustrated inFIG. 5-10A and FIG. 5-10B, for which two charge-storage regions are usedto acquire fluorescent emission from the reaction chambers. Thehistogram's bins can indicate a number of photons detected during eachtime interval after excitation of the fluorophore(s) in a reactionchamber 5-330. In some embodiments, signals for the bins will beaccumulated following a large number of excitation pulses, as depictedin FIG. 5-10A. The excitation pulses can occur at times t_(e1), t_(e2),t_(e3), . . . t_(eN) which are separated by the pulse interval time T.In some cases, there can be between 105 and 107 excitation pulses 5-122(or portions thereof) applied to a reaction chamber during anaccumulation of signals in the electron-storage regions for a singleevent being observed in the reaction chamber (e.g., a single nucleotideincorporation event in DNA analysis). In some embodiments, one bin (bin0) can be configured to detect an amplitude of excitation energydelivered with each optical pulse, and may be used as a reference signal(e.g., to normalize data). In other cases, the excitation pulseamplitude may be stable, determined one or more times during signalacquisition, and not determined after each excitation pulse so thatthere is no bin0 signal acquisition after each excitation pulse. In suchcases, carriers produced by an excitation pulse can be rejected anddumped from the photon-absorption/carrier-generation region 5-902 asdescribed above in connection with FIG. 5-9 .

In some implementations, only a single photon may be emitted from afluorophore following an excitation event, as depicted in FIG. 5-10A.After a first excitation event at time t_(e1), the emitted photon attime to may occur within a first time interval (e.g., between times t₁and t₂), so that the resulting electron signal is accumulated in thefirst electron-storage region (contributes to bin 1). In a subsequentexcitation event at time t_(e2), the emitted photon at time t_(f2) mayoccur within a second time interval (e.g., between times t₂ and t₃), sothat the resulting electron signal contributes to bin 2. After a nextexcitation event at time t_(e3), a photon may emit at a time t_(f3)occurring within the first time interval.

In some implementations, there may not be a fluorescent photon emittedand/or detected after each excitation pulse received at a reactionchamber 5-330. In some cases, there can be as few as one fluorescentphoton that is detected at a reaction chamber for every 10,000excitation pulses delivered to the reaction chamber. One advantage ofimplementing a mode-locked laser 5-113 as the pulsed excitation source5-106 is that a mode-locked laser can produce short optical pulseshaving high intensity and quick turn-off times at high pulse-repetitionrates (e.g., between 50 MHz and 250 MHz). With such highpulse-repetition rates, the number of excitation pulses within a 10millisecond charge-accumulation interval can be 50,000 to 250,000, sothat detectable signal can be accumulated.

After a large number of excitation events and carrier accumulations, thecarrier-storage regions of the time-binning photodetector 5-322 can beread out to provide a multi-valued signal (e.g., a histogram of two ormore values, an N-dimensional vector, etc.) for a reaction chamber. Thesignal values for each bin can depend upon the decay rate of thefluorophore. For example and referring again to FIG. 5-8 , a fluorophorehaving a decay curve B will have a higher ratio of signal in bin 1 tobin 2 than a fluorophore having a decay curve A. The values from thebins can be analyzed and compared against calibration values, and/oreach other, to determine the particular fluorophore present. For asequencing application, identifying the fluorophore can determine thenucleotide or nucleotide analog that is being incorporated into agrowing strand of DNA, for example. For other applications, identifyingthe fluorophore can determine an identity of a molecule or specimen ofinterest, which may be linked to the fluorophore.

To further aid in understanding the signal analysis, the accumulated,multi-bin values can be plotted as a histogram, as depicted in FIG.5-10B for example, or can be recorded as a vector or location inN-dimensional space. Calibration runs can be performed separately toacquire calibration values for the multi-valued signals (e.g.,calibration histograms) for four different fluorophores linked to thefour nucleotides or nucleotide analogs. As an example, the calibrationhistograms may appear as depicted in FIG. 5-11A (fluorescent labelassociated with the T nucleotide), FIG. 5-11B (fluorescent labelassociated with the A nucleotide), FIG. 5-11C (fluorescent labelassociated with the C nucleotide), and FIG. 5-11D (fluorescent labelassociated with the G nucleotide). A comparison of the measuredmulti-valued signal (corresponding to the histogram of FIG. 5-10B) tothe calibration multi-valued signals can determine the identity “T”(FIG. 5-11A) of the nucleotide or nucleotide analog being incorporatedinto the growing strand of DNA.

In some implementations, fluorescent intensity can be used additionallyor alternatively to distinguish between different fluorophores. Forexample, some fluorophores may emit at significantly differentintensities or have a significant difference in their probabilities ofexcitation (e.g., at least a difference of about 35%) even though theirdecay rates may be similar. By referencing binned signals (bins 5-3) tomeasured excitation energy and/or other acquired signals, it can bepossible to distinguish different fluorophores based on intensitylevels.

In some embodiments, different numbers of fluorophores of the same typecan be linked to different nucleotides or nucleotide analogs, so thatthe nucleotides can be identified based on fluorophore intensity. Forexample, two fluorophores can be linked to a first nucleotide (e.g.,“C”) or nucleotide analog and four or more fluorophores can be linked toa second nucleotide (e.g., “T”) or nucleotide analog. Because of thedifferent numbers of fluorophores, there may be different excitation andfluorophore emission probabilities associated with the differentnucleotides. For example, there may be more emission events for the “T”nucleotide or nucleotide analog during a signal accumulation interval,so that the apparent intensity of the bins is significantly higher thanfor the “C” nucleotide or nucleotide analog.

Distinguishing nucleotides or any other biological or chemical specimensbased on fluorophore decay rates and/or fluorophore intensities enablesa simplification of the optical excitation and detection systems in ananalytical instrument 5-100. For example, optical excitation can beperformed with a single-wavelength source (e.g., a source producing onecharacteristic wavelength rather than multiple sources or a sourceoperating at multiple different characteristic wavelengths).Additionally, wavelength-discriminating optics and filters may not beneeded in the detection system to distinguish between fluorophores ofdifferent wavelengths. Also, a single photodetector can be used for eachreaction chamber to detect emission from different fluorophores.However, in some embodiments, it may be advantageous to add additionaldimensions of discrimination for identifying a particular molecule byusing multiple of intensity, lifetime, wavelength, pulse duration and/orinterpulse duration to distinguish a sample.

The phrase “characteristic wavelength” or “wavelength” is used to referto a central or predominant wavelength within a limited bandwidth ofradiation (e.g., a central or peak wavelength within a 20 nm bandwidthoutput by a pulsed optical source). In some cases, “characteristicwavelength” or “wavelength” may be used to refer to a peak wavelengthwithin a total bandwidth of radiation output by a source.

Fluorophores having emission wavelengths in a range between about 560 nmand about 900 nm can provide adequate amounts of fluorescence to bedetected by a time-binning photodetector (which can be fabricated on asilicon wafer using CMOS processes). These fluorophores can be linked tobiological molecules of interest, such as nucleotides or nucleotideanalogs for genetic sequencing applications. Fluorescent emission inthis wavelength range can be detected with higher responsivity in asilicon-based photodetector than fluorescence at longer wavelengths.Additionally, fluorophores and associated linkers in this wavelengthrange may not interfere with incorporation of the nucleotides ornucleotide analogs into growing strands of DNA. In some implementations,fluorophores having emission wavelengths in a range between about 560 nmand about 660 nm can be optically excited with a single-wavelengthsource. An example fluorophore in this range is Alexa Fluor 647,available from Thermo Fisher Scientific Inc. of Waltham, Massachusetts.Excitation energy at shorter wavelengths (e.g., between about 500 nm andabout 650 nm) may be used to excite fluorophores that emit atwavelengths between about 560 nm and about 900 nm. In some embodiments,the time-binning photodetectors can efficiently detect longer-wavelengthemission from the reaction chambers, e.g., by incorporating othermaterials, such as Ge, into the photodetectors' active regions.

b. Protein Sequencing Applications

Some aspects of the present disclosure may be useful for proteinsequencing. For example, some aspects of the present disclosure areuseful for determining amino acid sequence information from polypeptides(e.g., for sequencing one or more polypeptides) such as by applying themulti-dimensional discrimination techniques using wavelength, lifetime,intensity, pulse duration and/or interpulse duration measurements toidentify a particular sample. In some embodiments, amino acid sequenceinformation can be determined for single polypeptide molecules. In someembodiments, one or more amino acids of a polypeptide are labeled (e.g.,directly or indirectly) and the relative positions of the labeled aminoacids in the polypeptide are determined. In some embodiments, therelative positions of amino acids in a protein are determined using aseries of amino acid labeling and cleavage steps. In particular, themulti-dimensional discrimination techniques described herein may beimplemented with the protein sequencing methods described in U.S. patentapplication Ser. No. 16/686,028 titled “METHODS AND COMPOSITIONS FORPROTEIN SEQUENCING,” filed Nov. 15, 2019 under Attorney Docket No.R0708.70042US02 and PCT Application No. PCT/US19/61831 titled “METHODSAND COMPOSITIONS FOR PROTEIN SEQUENCING,” filed Nov. 15, 2019 underAttorney Docket No. R0708.70042WO00, both which are hereby incorporatedby reference in their entireties

For example, FIG. 5-12 illustrates an example graph illustrating athree-dimensional discrimination technique for identifying amino acids.The inventors have recognized that it is possible to identify individualamino acids based at least one the characteristic intensity, pulseduration, and lifetime measurements. In particular, by obtainingmeasurements of intensity, pulse duration, and lifetime of emitted lightfrom a fluorescent marker, the obtained measurements can be compared toknown measurements to determine the most likely fluorescent marker fromwhich the emission light came from. By identifying a particularfluorescent marker, the amino acid to which the marker is attached canthen be known. Using three dimensions of discrimination, in particular,intensity, pulse duration, and lifetime, it is possible to identify alltwenty standard genetic amino acids as well as post-translationalmodifications. Although in the illustrated embodiment, athree-dimensional discrimination technique is illustrated foridentifying particular amino acids, the inventors have appreciated thatany of the multi-dimensional techniques may be applied to proteinsequencing application, including two-dimensional, four-dimensional,and/or five-dimensional techniques. In addition, although, in theillustrated embodiments, the three-dimensional discrimination techniqueis shown using measurements of intensity, lifetime, and pulse duration,it should be appreciated that other suitable combinations may beimplemented in the multi-dimensional techniques described herein, forexample, any of intensity, lifetime, wavelength, pulse duration and/orinterpulse duration.

In some embodiments, the identity of a terminal amino acid (e.g., anN-terminal or a C-terminal amino acid) is assessed, after which theterminal amino acid is removed and the identity of the next amino acidat the terminus is assessed, and this process is repeated until aplurality of successive amino acids in the polypeptide are assessed. Insome embodiments, assessing the identity of an amino acid comprisesdetermining the type of amino acid that is present. In some embodiments,determining the type of amino acid comprises determining the actualamino acid identity, for example by determining which of thenaturally-occurring 20 amino acids is the terminal amino acid is (e.g.,using a recognition molecule that is specific for an individual terminalamino acid). However, in some embodiments assessing the identity of aterminal amino acid type can comprise determining a subset of potentialamino acids that can be present at the terminus of the polypeptide. Insome embodiments, this can be accomplished by determining that an aminoacid is not one or more specific amino acids (and therefore could be anyof the other amino acids). In some embodiments, this can be accomplishedby determining which of a specified subset of amino acids (e.g., basedon size, charge, hydrophobicity, binding properties) could be at theterminus of the polypeptide (e.g., using a recognition molecule thatbinds to a specified subset of two or more terminal amino acids).

Amino acids of a polypeptide can be indirectly labeled, for example,using amino acid recognition molecules that selectively bind one or moretypes of amino acids on the polypeptide. Amino acids of a polypeptidecan be directly labeled, for example, by selectively modifying one ormore types of amino acid side chains on the polypeptide with uniquelyidentifiable labels. Methods of selective labeling of amino acid sidechains and details relating to the preparation and analysis of labeledpolypeptides are known in the art (see, e.g., Swaminathan, et al. PLoSComput Biol. 2015, 11(2):e1004080). Accordingly, in some embodiments,the one or more types of amino acids are identified by detecting bindingof one or more amino acid recognition molecules that selectively bindthe one or more types of amino acids. In some embodiments, the one ormore types of amino acids are identified by detecting labeledpolypeptide.

In some embodiments, the relative position of labeled amino acids in aprotein can be determined without removing amino acids from the proteinbut by translocating a labeled protein through a pore (e.g., a proteinchannel) and detecting a signal (e.g., a Förster resonance energytransfer (FRET) signal) from the labeled amino acid(s) duringtranslocation through the pore in order to determine the relativeposition of the labeled amino acids in the protein molecule.

As used herein, sequencing a polypeptide refers to determining sequenceinformation for a polypeptide. In some embodiments, this can involvedetermining the identity of each sequential amino acid for a portion (orall) of the polypeptide. However, in some embodiments, this can involveassessing the identity of a subset of amino acids within the polypeptide(e.g., and determining the relative position of one or more amino acidtypes without determining the identity of each amino acid in thepolypeptide). However, in some embodiments amino acid contentinformation can be obtained from a polypeptide without directlydetermining the relative position of different types of amino acids inthe polypeptide. The amino acid content alone may be used to infer theidentity of the polypeptide that is present (e.g., by comparing theamino acid content to a database of polypeptide information anddetermining which polypeptide(s) have the same amino acid content).

In some embodiments, sequence information for a plurality of polypeptideproducts obtained from a longer polypeptide or protein (e.g., viaenzymatic and/or chemical cleavage) can be analyzed to reconstruct orinfer the sequence of the longer polypeptide or protein. Accordingly,some embodiments provide compositions and methods for sequencing apolypeptide by sequencing a plurality of fragments of the polypeptide.In some embodiments, sequencing a polypeptide comprises combiningsequence information for a plurality of polypeptide fragments toidentify and/or determine a sequence for the polypeptide. In someembodiments, combining sequence information may be performed by computerhardware and software. The methods described herein may allow for a setof related polypeptides, such as an entire proteome of an organism, tobe sequenced. In some embodiments, a plurality of single moleculesequencing reactions may be performed in parallel (e.g., on a singlechip). For example, in some embodiments, a plurality of single moleculesequencing reactions are each performed in separate sample wells on asingle chip.

In some embodiments, methods provided herein may be used for thesequencing and identification of an individual protein in a samplecomprising a complex mixture of proteins. Some embodiments providemethods of uniquely identifying an individual protein in a complexmixture of proteins. In some embodiments, an individual protein isdetected in a mixed sample by determining a partial amino acid sequenceof the protein. In some embodiments, the partial amino acid sequence ofthe protein is within a contiguous stretch of approximately 5 to 50amino acids.

Without wishing to be bound by any particular theory, it is believedthat most human proteins can be identified using incomplete sequenceinformation with reference to proteomic databases. For example, simplemodeling of the human proteome has shown that approximately 98% ofproteins can be uniquely identified by detecting just four types ofamino acids within a stretch of 6 to 40 amino acids (see, e.g.,Swaminathan, et al. PLoS Comput Biol. 2015, 11(2):e1004080; and Yao, etal. Phys. Biol. 2015, 12(5):055003). Therefore, a complex mixture ofproteins can be degraded (e.g., chemically degraded, enzymaticallydegraded) into short polypeptide fragments of approximately 6 to 40amino acids, and sequencing of this polypeptide library would reveal theidentity and abundance of each of the proteins present in the originalcomplex mixture. Compositions and methods for selective amino acidlabeling and identifying polypeptides by determining partial sequenceinformation are described in in detail in U.S. patent application Ser.No. 15/510,962, filed Sep. 15, 2015, titled “SINGLE MOLECULE PEPTIDESEQUENCING,” which is hereby incorporated by reference in its entirety.

Sequencing in accordance with some embodiments can involve immobilizinga polypeptide on a surface of a substrate or solid support, such as achip or integrated device. In some embodiments, a polypeptide can beimmobilized on a surface of a sample well (e.g., on a bottom surface ofa sample well) on a substrate. In some embodiments, a first terminus ofa polypeptide is immobilized to a surface, and the other terminus issubjected to a sequencing reaction as described herein. For example, insome embodiments, a polypeptide is immobilized to a surface through aC-terminal end, and terminal amino acid recognition and degradationproceeds from an N-terminal end of the polypeptide toward the C-terminalend. In some embodiments, the N-terminal amino acid of the polypeptideis immobilized (e.g., attached to the surface). In some embodiments, theC-terminal amino acid of the polypeptide is immobilized (e.g., attachedto the surface). In some embodiments, one or more non-terminal aminoacids are immobilized (e.g., attached to the surface). The immobilizedamino acid(s) can be attached using any suitable covalent ornon-covalent linkage, for example as described herein. In someembodiments, a plurality of polypeptides are attached to a plurality ofsample wells (e.g., with one polypeptide attached to a surface, forexample a bottom surface, of each sample well), for example in an arrayof sample wells on a substrate.

Some aspects of the present disclosure provide a method of sequencing apolypeptide by detecting luminescence of a labeled polypeptide which issubjected to repeated cycles of terminal amino acid modification andcleavage. For example, FIG. 5-13 shows a method of sequencing a labeledpolypeptide by Edman degradation in accordance with some embodiments. Insome embodiments, the method generally proceeds as described herein forother methods of sequencing by Edman degradation. For example, in someembodiments, steps (1) and (2) shown in FIG. 5-13 may be performed asdescribed elsewhere herein for terminal amino acid modification andterminal amino acid cleavage, respectively, in an Edman degradationreaction.

As shown in the example depicted in FIG. 5-13 , in some embodiments, themethod comprises a step of (1) modifying the terminal amino acid of alabeled polypeptide. As described elsewhere herein, in some embodiments,modifying comprises contacting the terminal amino acid with anisothiocyanate (e.g., PITC) to form an isothiocyanate-modified terminalamino acid. In some embodiments, an isothiocyanate modification 5-1310converts the terminal amino acid to a form that is more susceptible toremoval by a cleaving reagent (e.g., a chemical or enzymatic cleavingreagent, as described herein). Accordingly, in some embodiments, themethod comprises a step of (2) removing the modified terminal amino acidusing chemical or enzymatic means including as detailed elsewhere hereinfor Edman degradation.

In some embodiments, the method comprises repeating steps (1) through(2) for a plurality of cycles, during which luminescence of the labeledpolypeptide is detected, and cleavage events corresponding to theremoval of a labeled amino acid from the terminus may be detected as adecrease in detected signal. In some embodiments, no change in signalfollowing step (2) as shown in FIG. 5-13 identifies an amino acid ofunknown type. Accordingly, in some embodiments, partial sequenceinformation may be determined by evaluating a signal detected followingstep (2) during each sequential round by assigning an amino acid type bya determined identity based on a change in detected signal oridentifying an amino acid type as unknown based on no change in adetected signal.

Some aspects of the present disclosure provide methods of polypeptidesequencing in real-time by evaluating binding interactions of terminalamino acids with labeled amino acid recognition molecules and a labeledcleaving reagent (e.g., a labeled exopeptidase). FIG. 5-14 shows anexample of a method of sequencing in which discrete binding events giverise to signal pulses of a signal output 5-1400. The inset panel of FIG.5-14 illustrates a general scheme of real-time sequencing by thisapproach. As shown, a labeled amino acid recognition molecule 5-1410selectively binds to and dissociates from a terminal amino acid (shownhere as lysine), which gives rise to a series of pulses in signal output5-1400 which may be used to identify the terminal amino acid. In someembodiments, the series of pulses provide a pulsing pattern which may bediagnostic of the identity of the corresponding terminal amino acid.

Without wishing to be bound by theory, labeled amino acid recognitionmolecule 5-1410 selectively binds according to a binding affinity (KD)defined by an association rate of binding (kon) and a dissociation rateof binding (koff). The rate constants koff and kon are the criticaldeterminants of pulse duration (e.g., the time corresponding to adetectable binding event) and interpulse duration (e.g., the timebetween detectable binding events), respectively. In some embodiments,these rates can be engineered to achieve pulse durations and pulse ratesthat give the best sequencing accuracy.

As shown in the inset panel, a sequencing reaction mixture furthercomprises a labeled cleaving reagent 5-1420 comprising a detectablelabel that is different than that of labeled amino acid recognitionmolecule 5-1410. In some embodiments, labeled cleaving reagent 5-1420 ispresent in the mixture at a concentration that is less than that oflabeled amino acid recognition molecule 5-1410. In some embodiments,labeled cleaving reagent 5-1420 displays broad specificity such that itcleaves most or all types of terminal amino acids.

As illustrated by the progress of signal output 5-1400, in someembodiments, terminal amino acid cleavage by labeled cleaving reagent5-1420 gives rise to a uniquely identifiable signal pulse, and theseevents occur with higher wavelength than the binding pulses of a labeledamino acid recognition molecule 5-1410. In this way, amino acids of apolypeptide can be counted and/or identified in a real-time sequencingprocess. As further illustrated in signal output 5-1400, in someembodiments, a labeled amino acid recognition molecule 5-1410 isengineered to bind more than one type of amino acid with differentbinding properties corresponding to each type, which produces uniquelyidentifiable pulsing patterns. In some embodiments, a plurality oflabeled amino acid recognition molecules may be used, each with adiagnostic pulsing pattern, including a characteristic wavelength,lifetime, intensity, pulse duration and/or interpulse duration, whichmay be used to identify a corresponding terminal amino acid.

VIII. Alternatives and Scope

Having thus described several aspects and embodiments of the technologyof the present disclosure, it is to be appreciated that variousalterations, modifications, and improvements will readily occur to thoseof ordinary skill in the art. Such alterations, modifications, andimprovements are intended to be within the spirit and scope of thetechnology described herein. It is, therefore, to be understood that theforegoing embodiments are presented by way of example only and that,within the scope of the appended claims and equivalents thereto,inventive embodiments may be practiced otherwise than as specificallydescribed. In addition, any combination of two or more features,systems, articles, materials, kits, and/or methods described herein, ifsuch features, systems, articles, materials, kits, and/or methods arenot mutually inconsistent, is included within the scope of the presentdisclosure.

Also, as described, some aspects may be embodied as one or more methods.The acts performed as part of the method may be ordered in any suitableway. Accordingly, embodiments may be constructed in which acts areperformed in an order different than illustrated, which may includeperforming some acts simultaneously, even though shown as sequentialacts in illustrative embodiments.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. The transitional phrases “consisting of” and “consisting essentiallyof” shall be closed or semi-closed transitional phrases, respectively.

What is claimed is: 1-20. (canceled)
 21. A method comprising:generating, with at least one photodetection region of an integratedcircuit, charge carriers responsive to incident photons emitted from asample; obtaining information about the incident photons, theinformation comprising pulse duration information and/or interpulseduration information and at least one member selected from a groupcomprising wavelength information, luminescence lifetime information,and intensity information; wherein: the pulse duration information isobtained at least in part by determining a first pulse comprisingmeasures of arrivals of a plurality of incident photons emitted from thesample and determining a length of a first time interval during whichthe first pulse exceeds a first threshold; and the interpulse durationinformation is obtained at least in part by determining a second pulsecomprising measures of arrivals of a second plurality of incidentphotons emitted from the sample and determining a length of a timeinterval between the first time interval and a second time intervalduring which the second pulse exceeds a second threshold.
 22. The methodof claim 21, further comprising receiving, with at least one chargestorage region of the integrated circuit, the charge carriers from theat least one photodetection region.
 23. The method of claim 21, furthercomprising identifying the sample based at least in part on theinformation obtained.
 24. The method of claim 23, wherein identifyingthe sample comprises identifying at least one fluorescent marker and atleast one molecule to which the at least one fluorescent marker isattached.
 25. The method of claim 25, wherein identifying the at leastone molecule comprises identifying at least one amino acid.
 26. Themethod of claim 21, wherein the information comprises at least twomembers selected from the group comprising wavelength information,luminescence lifetime information, and intensity information.
 27. Themethod of claim 21, wherein the information comprises wavelengthinformation, luminescence lifetime information, and intensityinformation.
 28. A system comprising: at least one chamber for receivinga sample; and at least one component configured to obtain informationabout incident photons emitted from the sample in response to excitationlight, the information comprising pulse duration information and/orinterpulse duration information and at least one member selected from agroup comprising wavelength information, luminescence lifetimeinformation, and intensity information; wherein: the pulse durationinformation is obtained at least in part by determining a first pulsecomprising measures of arrivals of a plurality of incident photonsemitted from the sample and determining a length of a first timeinterval during which the first pulse exceeds a first threshold; and theinterpulse duration information is obtained at least in part bydetermining a second pulse comprising measures of arrivals of a secondplurality of incident photons emitted from the sample and determining alength of a time interval between the first time interval and a secondtime interval during which the second pulse exceeds a second threshold.29. The system of claim 28, further comprising: at least onephotodetection region configured to generate charge carriers responsiveto the incident photons emitted from the sample; and at least one chargestorage region configured to receive the charge carriers from thephotodetection region.
 30. The system of claim 28, wherein the at leastone chamber comprises a plurality of chambers, each of the plurality ofchambers configured to receive a portion of the sample.
 31. The systemof claim 28, wherein the system is configured to identify the samplebased at least in part on the information obtained by the at least onecomponent.
 32. The system of claim 28, wherein the information comprisesat least two members selected from the group comprising wavelengthinformation, luminescence lifetime information, and intensityinformation.
 33. The system of claim 28, wherein the informationcomprises wavelength information, luminescence lifetime information, andintensity information.
 34. The system of claim 28, further comprising anintegrated device comprising the at least one chamber.
 35. A systemcomprising: at least one chamber for receiving a sample; and at leastone component configured to determine information about the incidentphotons, the information comprising at least three members selected froma group comprising a characteristic wavelength of the incident photonsemitted from the sample, a characteristic luminescence lifetime of oneor more of the incident photons emitted from the sample, acharacteristic intensity of the sample, a characteristic pulse durationof the sample, and a characteristic interpulse duration of the sample;wherein: the system is configured to identify the sample at least inpart by comparing the information of each of the three membersdetermined by the at least one component to known information about acandidate matching sample.
 36. The system of claim 35, furthercomprising: at least one photodetection region configured to generatecharge carriers responsive to the incident photons emitted from thesample; and at least one charge storage region configured to receive thecharge carriers from the photodetection region.
 37. The system of claim35, wherein the at least one chamber comprises a plurality of chambers,each of the plurality of chambers configured to receive a portion of thesample.
 38. The system of claim 35, wherein the information determinedby the at least one component comprises at least two members selectedfrom a group comprising the characteristic wavelength of the incidentphotons emitted from the sample, the characteristic luminescencelifetime of the one or more of the incident photons emitted from thesample, and the characteristic intensity of the sample, and one memberselected from a group comprising the characteristic pulse duration ofthe sample and the characteristic interpulse duration of the sample. 39.The system of claim 35, wherein the information obtained by the at leastone component comprises the characteristic luminescence lifetime of theone or more of the incident photons emitted from the sample, thecharacteristic intensity of the sample, and one member selected from thegroup comprising the characteristic pulse duration of the sample and thecharacteristic interpulse duration of the sample.
 40. The system ofclaim 35, further comprising an integrated device comprising the atleast one chamber.